Design of novel analogue peptides with potent antibiotic activity based on the antimicrobial peptide,HP (2-20), derived from N-terminus of Helicobacter pylori ribosomal protein L1

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RPL1). In order to develop novel antibiotic peptides useful as therapeutic agents, potent antibiotic activities against bacteria, fungi and cancer cells without a cytotoxic effect are essential. To this end, several analogues with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, the substitution of Trp for the hydrophobic amino acids, Gln and Asp at positions 17 and 19 of HP (2-20) (Anal 3), caused a dramatic increase in antibiotic activity without a hemolytic effect.In contrast, the decrease of hydrophobicity brought about by substituting Ser for Leu and Phe at positions 12 and 19 of HP (2-20), respectively (Anal 4, Anal 5), did not have a significant effect on the antibiotic activity. The antibiotic effects of these synthetic peptides were further investigated by treating prepared protoplasts of Candida albicans and conducting an artificial liposomal vesicle (PC/PS; 3:1, w/w) disrupting activity test. The results demonstrated that the Anal 3 prevented the regeneration of fungal cell walls and induced an enhanced release of fluorescent dye (carboxyfluorescein) trapped in the artificial membrane vesicles to a greater degree than HP (2-20).The potassium-release test conducted on C. albicans indicated that Anal 3 induced greater amounts of potassium ion to be released than the parent peptide, HP (2-20) did. These results indicated that the hydrophobic region of peptides is prerequisite for its effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.     

Prevention of lethal murine candidiasis using HP (2-20), an antimicrobial peptide derived from the N-terminus of Helicobacter pylori ribosomal protein L1

Peptide HP (2-20), A(2)KKVFKRLEKLFSKIQNDK(20), is a cationic antimicrobial peptide derived from the N-terminus of Helicobacter pylori ribosomal protein 1, HpRpL1. Native peptide HP (2-20) and its synthetic derivatives have been shown in vitro to exhibit potent killing activity against Gram-positive, Gram-negative and yeast cells, thus, making them promising candidates for treatment of polymicrobial infections. However, the therapeutic potential of peptide HP (2-20) or its synthetic derivatives in any animal model of either bacterial or fungal diseases has not yet been investigated. In this study, we demonstrate that synthetic peptide amide HP (2-20), administered in six doses (300microg each; one intraperitoneal dose at the time of the infection, followed by five intravenous doses at 12h intervals) to CBA/J male mice experimentally infected with a lethal inoculum ( [Formula: see text] CFU) of Candida albicans, delayed the onset of disease, suppressed disease progression, and greatly increased survival rate and time (16.6% by day 14), as compared with the untreated infected control mice (100% mortality by day 5). Further, using isotonic buffer systems differing in ionic strength, peptide HP (2-20) was shown in vitro to exhibit an ionic strength-dependent hemolytic activity, previously not detected. Repeated intravenous administration of uninfected control CBA/J male mice with peptide HP (2-20), however, caused neither morbidity nor mortality. These findings strongly evidence the therapeutic efficacy and safety values of peptide HP (2-20) as a lead drug for the treatment of acquired candidiasis.     

Antifungal mechanism of an antimicrobial peptide, HP (2--20), derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans

The antifungal activity and mechanism of HP (2-20), a peptide derived from the N-terminus sequence of Helicobacter pylori Ribosomal Protein L1 were investigated. HP (2--20) displayed a strong antifungal activity against various fungi, and the antifungal activity was inhibited by Ca(2+) and Mg(2+) ions. In order to investigate the antifungal mechanism(s) of HP (2-20), fluorescence activated flow cytometry was performed. As determined by propidium iodide staining, Candida albicans treated with HP (2-20) showed a higher fluorescence intensity than untreated cells and was similar to melittin-treated cells. The effect on fungal cell membranes was examined by investigating the change in membrane dynamics of C. albicans using 1,6-diphenyl-1,3,5-hexatriene as a membrane probe and by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w) and by treating protoplasts of C. albicans with the peptide. The action of peptide against fungal cell membrane was further examined by the potassium-release test, and HP (2-20) was able to increase the amount of K(+) released from the cells. The result suggests that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membrane via pore formation or directly interacts with the lipid bilayers in a salt-dependent manner.     

Amphipathic alpha-helical peptide, HP (2-20), and its analogues derived from Helicobacter pylori: pore formation mechanism in various lipid compositions

In a previous study, we determined that HP(2-20) (residues 2-20 of parental HP derived from the N-terminus of Helicobacter pylori Ribosomal Protein L1) and its analogue, HPA3, exhibit broad-spectrum antimicrobial activity. The primary objective of the present study was to gain insight into the relevant mechanisms of action using analogues of HP(2-20) together with model liposomes of various lipid compositions and electron microscopy. We determined that these analogues, HPA3 and HPA3NT3, exert potent antibacterial effects in low-salt buffer and antifungal activity against chitin-containing fungi, while having little or no hemolytic activity or cytotoxicity against mammalian cell lines. Our examination of the interaction of HP(2-20) and its analogues with liposomes showed that the peptides disturb both neutral and negatively-charged membranes, as demonstrated by the release of encapsulated fluorescent markers. The release of fluorescent markers induced by HP(2-20) and its analogues was inversely related to marker size. The pore created by HP(2-20) shows that the radius is approximately 1.8 nm, whereas HPA3, HPA3NT3, and melittin have apparent radii between 3.3 and 4.8 nm. Finally, as shown by electron microscopy, the liposomes and various microbial cells treated with HPA3 and HPA3NT3 showed oligomerization and blebbing similar to that seen with melittin, while HP(2-20) exhibited flabbiness. These results suggest that HP(2-20) may exert its antibiotic effects through a small pore (about 1.8 nm), whereas HPA3 and HPA3NT3 formed pores of a size consistent with those formed by melittin.     

The role played by lipids unsaturation upon the membrane interaction of the Helicobacter pylori HP(2–20) antimicrobial peptide analogue HPA3

The HPA3 peptide is an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein, able to interact with zwitterionic lipid membranes and generate pores. Herein we focused on the importance of the degree of unsaturation of lipid acyl chains on HPA3 peptide-membrane interactions. Electrophysiology experiments carried out in reconstituted lipid membranes formed from phosphatidylcholines with one (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine − POPC) and two monounsaturated acyl chains (1,2-dioleoyl-sn-glycero-3-phosphocholine − DOPC) demonstrate that the lesser degree of the packing density of membrane lipids encountered in DOPC-based planar membranes greatly enhances the electric activity of pores created by the HPA3 peptide. Data derived from fluorescence spectroscopy experiments demonstrate that upon interaction with the bilayer, the HPA3 peptide translocates to the trans-side of the membrane. From the same experiments, we demonstrate that in the case of DOPC-based planar membranes, the net amount of HPA3 peptide which passes across the membrane and re-dissolves in the trans solution is almost 22% greater than POPC-based membranes. Such data further emphasize the modulatory role played by lipid acyl chain in determining antimicrobial peptides-lipids interactions, and demonstrate that small differences in unsaturation degree can impose a sizeable influence on HPA3 peptide activity.     

Surface localized Heat Shock Protein 20 (HslV) of Helicobacter pylori

Background. Heat Shock Protein (HSP) has been regarded as a pathogenic factor in Helicobacter pylori infection. Heat Shock Protein 20 (HSP20) of H. pylori is identified as Hs1V based on open reading frame predication of genome sequences. It is a homologue of HslV of E. coli, a peptidase involved in protein degradation. Methods. The HSP20 gene was cloned and inserted into pET16b fused with His‐tag. Recombinant HSP20 protein (rHSP20) was expressed and purified by nickel column. Rabbit anti‐rHSP20 was purified by Protein A affinity chromatography and used as a probe to localize HSP20 in H. pylori by immuno‐gold labeling and Western blotting. rHSP20 was also used as antigen to test for antibody against HSP20 in patients with H. pylori infection by enzyme‐linked immunosorbant assay. Results. Immuno‐gold labeled transmission electron microscopy shows that HSP20 is located on the cell surface of H. pylori. Western blotting of 2‐D gel shows that HSP20 has a pI of ～5.5 and a molecular weight of ～18 kDa. The ELISA result shows that there is no significant difference in antibody titre against rHSP20 in all sera tested. Conclusion. The presence of IgG to rHSP20 may imply an earlier exposure of the patients and normal subjects to H. pylori. However, the mechanism has not been established. HSP20 has been shown to localize on the surface of H. pylori. Surface localization of H. pylori HSP20 may provide the path to a better understanding of the role and function of HSP20 in bacteria–host interaction.     

Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12)

To develop novel antibiotic peptides useful as therapeutic drugs, a number of analogues were designed to increase the hydrophobic helix region either by Trp-substitution or net positive charge increase by Lys-substitution, from HP(2-9)-ME(1-12). The antibiotic activities of these peptides were evaluated using bacterial (Salmonella tryphimurium, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus), fungi (Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans), tumor and human erythrocyte cells. The substitution of Lys for Thr at position 18 and 19 of HP(2-9)-ME(1-12) (HM5) increased activity against Proteus vulgaris and fungal strains without hemolysis. In contrast, substitution of Trp for Lys and Thr at positions 2, 15 and 19 of HP(2-9)-ME(1-12), respectively (HM3 and HM4), decreased activity but increased hemolysis against human erythrocytes. This suggests that an increase in positive charge increases antimicrobial activity whereas an increase in hydrophobicity by introducing Trp residues at C-terminus of HP(2-9)-ME(1-12) causes a hemolytic effect. Circular dichroism spectra suggested that the alpha-helical structure of these peptides plays an important role in their antibiotic effect but that the alpha-helical property is not connected with the enhanced antibiotic activity.     

Peptides with Dual Antimicrobial-Anticancer Activity Derived from the N-terminal Region of H. pylori Ribosomal Protein L1 (RpL1)

International Journal of Peptide Research and Therapeutics - Whereas the traditional approaches of cancer therapy including radiotherapy, chemotherapy, and immunotherapy have failed to properly...     

The crystal structure of ADP-L-glycero-D-manno-heptose-6-epimerase (HP0859) from Helicobacter pylori

Helicobacter pylori, the human pathogen that affects about half of the world population and that is responsible for gastritis, gastric ulcer and adenocarcinoma and MALT lymphoma, owes much of the integrity of its outer membrane on lipopolysaccharides (LPSs). Together with their essential structural role, LPSs contribute to the bacterial adherence properties, as well as they are well characterized for the capability to modulate the immuno-response. In H. pylori the core oligosaccharide, one of the three main domains of LPSs, shows a peculiar structure in the branching organization of the repeating units, which displayed further variability when different strains have been compared. We present here the crystal structure of ADP-L-glycero-D-manno-heptose-6-epimerase (HP0859, rfaD), the last enzyme in the pathway that produces L-glycero-D-manno-heptose starting from sedoheptulose-7-phosphate, a crucial compound in the synthesis of the core oligosaccharide. In a recent study, a HP0859 knockout mutant has been characterized, demonstrating a severe loss of lipopolysaccharide structure and a significant reduction of adhesion levels in an infection model to AGS cells, if compared with the wild type strain, in good agreement with its enzymatic role. The crystal structure reveals that the enzyme is a homo-pentamer, and NAD is bound as a cofactor in a highly conserved pocket. The substrate-binding site of the enzyme is very similar to that of its orthologue in Escherichia coli, suggesting also a similar catalytic mechanism.     

Design and characterization of novel hybrid peptides from LFB15(W4,10), HP(2-20), and cecropin A based on structure parameters by computer-aided method

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. The pivotal assets of the antimicrobial peptide include potential for rapid bactericidal activity and low propensity for resistance. The four new antimicrobial hybrid peptides were designed based on peptides LFB15(W4,10), HP(2-20), and cecropin A according to the structure–activity relationship of the amphipathic and cationic antimicrobial peptides. Their structural parameters were accessed by bioinformatics tools, and then two hybrids with the most potential candidates were synthesized. The hybrid peptide LH28 caused an increase in antibiotic activity (MIC₅₀ = 1.56–3.13 μM) against given bacterial strains and did not cause obvious hemolysis of rabbit erythrocytes at concentration of 3.13 μM with effective antimicrobial activity. The results demonstrate that evaluating the structural parameters could be useful for designing novel antimicrobial peptides. Zi-gang Tian and Tian-tang Dong contributed equally to this paper     

HP (2-20) derived from the amino terminal region of helicobacterpylori ribosomal protein L1 exerts its antifungal effects by damaging the plasma membranes of Candida albicans.

The fungicidal effects of the peptide HP (2-20). derived from the N-terminal sequence of Helicobacter pylori ribosomal protein L1 (RPL1). have been investigated. HP (2-20) displays a strong fungicidal activity against various fungi, without haemolytic activity against human erythrocyte cells, and the fungicidal activity is inhibited by Ca2+ and Mg2+ ions. In order to investigate the fungicidal mechanism(s) of HP (2-20). the amount of intracellular trehalose was measured in C. albicans. It was found that the amounts of intracellular trehalose were decreased when HP (2-20) was used. The action of the peptide against fungal cell membranes was further examined by the potassium-release test; HP (2-20) was found to increase the amount of K+ released from the cells. Furthermore, HP (2-20) caused significant morphological changes, as shown by scanning electron microscopy, and by testing the membrane disrupting activity using liposomes (phosphatidyl choline/cholesterol; 10: 1, w/w). Our results suggest that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membranes, via pore formation or direct interaction with the lipid bilayers.     

Convergent synthesis and cruzain inhibitory activity of novel 2-(N'-benzylidenehydrazino)-4-trifluoromethyl-pyrimidines

To search for new cruzain inhibitors, the synthesis of a series of novel 2-(N'-benzylidenehydrazino)-4-trifluoromethyl-pyrimidines in a convergent manner is presented. The cruzain inhibitory activity of some of these compounds was evaluated and a binding model was proposed. All derivatives tested were active and the most significant inhibitory effect (80% at 100 microM) and IC(50) value (85 microM) were obtained from the 2-(N'-4-chloro-benzylidenehydrazino)-4-trifluoromethyl-pyrimidine. Although further structural optimization to improve solubility is necessary, the molecular docking studies suggest that these inhibitors occupy the S2 pocket without irreversible enzyme inactivation, through hydrophobic interactions, thus, indicating a desirable mode of interaction for the design of novel inhibitors.     

A convenient synthesis of a novel nucleoside analogue: 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone

The nucleoside analogue 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone (5) was prepared from the corresponding 4-methyl pyrimidinone nucleoside by means of the Vilsmeier reaction. The unprotected nucleoside can be phosphorylated directly with phosphorus oxychloride in triethyl phosphate.     

Synthesis and CYP24A1 inhibitory activity of N-(2-(1H-imidazol-1-yl)-2-phenylethyl)arylamides

A series of N-(2-(1H-imidazol-1-yl)-2-phenylethyl)arylamides were prepared, using an efficient three- to five-step synthesis, and evaluated for their inhibitory activity against human cytochrome P450C24A1 (CYP24A1) hydroxylase. Inhibition ranged from IC₅₀ 0.3–72 μM compared with the standard ketoconazole IC₅₀ 0.52 μM, with the styryl derivative (11c) displaying enhanced activity (IC₅₀ = 0.3 μM) compared with the standard, providing a useful preliminary lead for drug development.     

Single-molecule investigation of the interactions between reconstituted planar lipid membranes and an analogue of the HP(2-20) antimicrobial peptide

In this study, we employed electrophysiology experiments carried out at the single-molecule level to study the mechanism of action of the HPA3 peptide, an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Amplitude analysis of currents fluctuations induced by HPA3 peptide at various potentials in zwitterionic lipid membranes reveal the existence of reproducible conductive states in the stochastic behavior of such events, which directly supports the existence of transmembrane pores induced the peptide. From our data recorded both at the single-pore and macroscopic levels, we propose that the HPA3 pore formation is electrophoretically facilitated by trans-negative transmembrane potentials, and HPA3 peptides translocate into the trans monolayers after forming the pores. We present evidence according to which the decrease in the membrane dipole potential of a reconstituted lipid membranes leads to an augmentation of the membrane activity of HPA3 peptides, and propose that a lower electric dipole field of the interfacial region of the membrane caused by phloretin facilitates the surface-bound HPA3 peptides to break free from one leaflet of the membrane, insert into the membrane and contribute to pore formation spanning the entire thickness of the membrane.     

Biosynthesis of a novel recombinant peptide derived from hPTH(1-34)

A recombinant human parathyroid hormone fragment, Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro (MW=4550 Da), was developed by substituting Arg for Leu at position 11 and adding Pro-Pro at the carboxyl terminus. Following a single injection (0.5-13.5 μg/bird) of the rhPTH fragment, the serum calcium level in chickens increased 12-42% (P<0.05) after 1h as determined by the Parson's Chicken Assay. The functional activity of Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro may be due to removal of the N-terminus Pro-Pro- by X-prolyl dipeptidyl peptidase IV (DPPIV) in vivo, increasing its activity compared to Pro-Pro-hPTH(1-34). This artificial rhPTH fragment could be used to increase calcium mobilization and potentially improve bone health.     

Negatively charged residues in the membrane ordering activity of SARS-CoV-1 and -2 fusion peptides

Entry of coronaviruses into host cells is mediated by the viral spike protein. Previously, we identified the bona fide fusion peptides (FPs) for severe acute respiratory syndrome coronavirus (“SARS-1”) and severe acute respiratory syndrome coronavirus-2 (“SARS-2”) using electron spin resonance spectroscopy. We also found that their FPs induce membrane ordering in a Ca²⁺-dependent fashion. Here we study which negatively charged residues in SARS-1 FP are involved in this binding, to build a topological model and clarify the role of Ca²⁺. Our systematic mutation study on the SARS-1 FP shows that all six negatively charged residues contribute to the FP’s membrane ordering activity, with D812 the dominant residue. The corresponding SARS-2 residue D830 plays an equivalent role. We provide a topological model of how the FP binds Ca²⁺ ions: its two segments FP1 and FP2 each bind one Ca²⁺. The binding of Ca²⁺, the folding of FP (both studied by isothermal titration calorimetry experiments), and the ordering activity correlate very well across the mutants, suggesting that the Ca²⁺ helps the folding of FP in membranes to enhance the ordering activity. Using a novel pseudotyped viral particle-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole spike protein in its trimer form in real time. We found that the SARS-1 and SARS-2 pseudotyped viral particles also induce membrane ordering to the extent that separate FPs do, and mutations of the negatively charged residues also significantly suppress the membrane ordering activity. However, the slower kinetics of the FP ordering activity versus that of the pseudotyped viral particle suggest the need for initial trimerization of the FPs.