Lipidated variants of the antimicrobial peptide nisin produced via incorporation of methionine analogs for click chemistry show improved bioactivity.

The increase in antibiotic resistance calls for accelerated molecular engineering strategies to diversify natural products for drug discovery. The incorporation of non-canonical amino acids (ncAAs) is an elegant strategy for this purpose, offering a diverse pool of building blocks to introduce desired properties into antimicrobial lanthipeptides. We here report an expression system using Lactococcus lactis as a host for non-canonical amino acid incorporation with high efficiency and yield. We show that incorporating the more hydrophobic analog ethionine (instead of methionine) into nisin improves its bioactivity against several Gram-positive strains we tested. New-to-nature variants were further created by click chemistry. By azidohomoalanine (Aha) incorporation and subsequent click chemistry, we obtained lipidated variants at different positions in nisin or in truncated nisin variants. Some of them show improved bioactivity and specificity against several pathogenic bacterial strains. These results highlight the ability of this methodology for lanthipeptide multi-site lipidation, to create new-to-nature antimicrobial products with diverse features, and extend the toolbox for (lanthi)peptide drug improvement and discovery.

Simplified lipid II-binding antimicrobial peptides: Design, synthesis and antimicrobial activity of bioconjugates of nisin rings A and B with pore-forming peptides.

New designs of antimicrobial peptides are urgently needed in order to combat the threat posed by the recent increase of resistance to antibiotics. In this paper, we present a new series of antimicrobial peptides, based on the key structural features of the lantibiotic nisin. We have simplified the structure of nisin by conjugating the lipid II-binding motif at the N-terminus of nisin to a series of cationic peptides and peptoids with known antibacterial action and pore-forming properties. Hybrid peptides, where a hydrophilic PEG4 linker was used, showed good antibacterial activity against Micrococcus luteus.

Incorporation of Nonproteinogenic Amino Acids in Class I and II Lantibiotics.

Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain thioether cross-links formed by lanthionine and methyllanthionine residues. They exert potent antimicrobial activity against Gram-positive bacteria. We herein report production of analogues of two lantibiotics, lacticin 481 and nisin, that contain nonproteinogenic amino acids using two different strategies involving amber stop codon suppression technology. These methods complement recent alternative approaches to incorporate nonproteinogenic amino acids into lantibiotics.

Preparation and characterization of aminoethyl hydroxypropyl methyl cellulose modified with nisin.

Nisin grafted aminoethyl hydroxypropyl methyl cellulose (AEHPMC) was prepared by an enzyme-catalyzed reaction in the presence of microbial transglutaminase (MTGase). AEHPMC was synthesized with 2-chloroethylamine hydrochloride (CEH) which was as an intermediate reactant. The parameters, which influenced the NH2% and the degree of substitution (DS), including reaction time, reaction temperature and the mass ratio of the reactants were investigated. Antioxidant activities of AEHPMC-nisin were evaluated by the scavenging activity of hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. With the DS and concentration increasing of AEHPMC-nisin, the radical scavenging activity increased. The Methylthiazol tetrazolium (MTT) assay indicated that AEHPMC-nisin had low-toxicity to L929 cells. Therefore, the derivative of HPMC may show a promising potential application in biomedical, food and pharmaceutical fields.

Synthesis of nisin AB dicarba analogs using ring-closing metathesis: influence of sp(3) versus sp(2) hybridization of the α-carbon atom of residues dehydrobutyrine-2 and dehydroalanine-5 on the lipid II binding affinity.

Herein the synthesis of two nisin AB dicarba analogs is described, focusing on amino acid modifications at positions 2 and 5. The nisin mimics were synthesized by a combination of solid phase synthesis of the linear peptides, followed by macrocyclization via ring-closing metathesis and fragment assembly by means of solution phase chemistry. The two N-terminal nisin AB-fragment mimics contain either the native dehydrobutyrine (Dhb)/dehydroalanine (Dha) amino acid residues or alanine at position 2 and 5, respectively. The native dehydrobutyrine at position 2 and dehydroalanine at position 5 were introduced as their precursors, namely threonine and serine, respectively, and subsequent dehydration was carried out by EDCI/CuCl as the condensing agent. Both AB-fragment mimics were analyzed in a lipid II binding assay and it was found that the Ala2/Ala5 AB-mimic (2) showed a reduced activity, while the Dhb2/Dha5 AB-mimic (3) was as active as the native AB-fragment (1).

Synthesis of antimicrobial Nisin-phosphorylated soybean protein isolate/poly(L-lactic acid)/ZrO2 membranes.

Electrospinning technique was used to fabricate the model drug Nisin loaded phosphorylated soybean protein isolate/poly(l-lactic acid)/zirconium dioxide (Nisin-PSPI/PLLA/ZrO2) nanofibrous membranes. The average diameter of drug carrier PSPI/PLLA/ZrO2 nanofibers increased with the increase of content PSPI and some spindle-shape beads appeared when PSPI content reached 25 wt%. The loading dosage of Nisin caused no significant changes in the size and morphology of nanofibers when Nisin content was below 9 wt%. There existed hydrogen and Zr-O-C bonds among PSPI, PLLA and ZrO2 units, and the crystalline of PLLA matrix decreased owning to the introducing of PSPI and ZrO2 units. Moreover, the water absorption capability and degradation rate of PSPI/PLLA/ZrO2 nanofibrous membranes increased with increasing PSPI content. The antimicrobial activity and release experimental results showed that Nisin-PSPI/PLLA/ZrO2 nanofibrous membranes displayed well controlled release and better antimicrobial activity against Staphylococcus aureus (S. aureus), and the Nisin release from the medicated nanofibers could be described by Fickian diffusion model. The Nisin-PSPI/PLLA/ZrO2 nanofibrous membranes may have potential as a new nanofibrous membrane in drug delivery, food active packaging and wound dressing.

Semi-synthesis of biologically active nisin hybrids composed of the native lanthionine ABC-fragment and a cross-stapled synthetic DE-fragment.

The antimicrobial peptide nisin is a promising template for designing novel peptide-based antibiotics to improve its drug-like properties. First steps in that direction represent the synthesis of hybrid nisin derivatives that contain a native nisin ABC-part and synthesized cross-stapled DE-ring fragments and are described here. The biological activity of the newly synthesized nisin derivatives was evaluated in order to compare the bioactivity of the synthetic DE-ring containing mimic and native lanthionine-bridged DE-ring containing nisin. The native nisin ABC-ring system was obtained via chymotrypsin digestion of full-length nisin, and was subsequently functionalized at the C-terminal carboxylate with two different amino alkyne moieties. Next, nisin hybrids were successfully prepared using Cu(I)-catalyzed azide alkyne cycloaddition 'click' chemistry by chemo-selective ligation of the ABC-alkyne with the N-terminal azido functionalized dicarba-DE ring mimic. The newly synthesized compounds were active as potent lipid II binders and retained antimicrobial activity in a growth inhibition assay. However, pore formation was not observed, possibly either due to the different character of the 'staples' as compared to the parent sulfides, or due to the triazole moiety as a sub-optimal amide bond isostere.

Synthesis, antimicrobial activity, and membrane permeabilizing properties of C-terminally modified nisin conjugates accessed by CuAAC.

Functionalization of the lantibiotic nisin with fluorescent reporter molecules is highly important for the understanding of its mode of action as a potent antimicrobial peptide. In addition to this, multimerization of nisin to obtain multivalent peptide constructs and conjugation of nisin to bioactive molecules or grafting it on surfaces can be attractive methods for interference with bacterial growth. Here, we report a convenient method for the synthesis of such nisin conjugates and show that these nisin derivatives retain both their antimicrobial activity and their membrane permeabilizing properties. The synthesis is based on the Cu(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) as a bioorthogonal ligation method for large and unprotected peptides in which nisin was C-terminally modified with propargylamine and subsequently efficiently conjugated to a series of functionalized azides. Two fluorescently labeled nisin conjugates together with a dimeric nisin construct were prepared while membrane insertion as well as antimicrobial activity were unaffected by these modifications. This study shows that C-terminal modification of nisin does not deteriorate biological activity in sharp contrast to N-terminal modification and therefore C-terminally modified nisin analogues are valuable tools to study the antibacterial mode of action of nisin. Furthermore, the ability to use stoichiometric amounts of the azide containing molecule opens up possibilities for surface tethering and more complex multivalent structures.

Synthesis and structural characterization of the individual diastereoisomers of a cross-stapled alkene-bridged nisin DE-ring mimic.

Herein, we describe the synthesis, structural characterization, and synthetic use as an advanced intermediate of a cross-stapled alkene-bridged hexapeptide to mimic the DE-ring of the lantibiotic nisin. The linear precursor was cyclized by ring-closing metathesis to give the correctly folded bicyclic hexapeptide in a single step, and the four individual diastereoisomers were isolated, structurally assigned and characterized by HPLC, NMR and MS, respectively. The bicyclic hexapeptide was used as a versatile advanced synthon and was modified at its C- and N-terminus, among others, with an azide moiety to access a building block suitable for Cu(I)-catalyzed alkyne-azide cycloaddition-based ligation reactions.

Synthesis of bicyclic alkene-/alkane-bridged nisin mimics by ring-closing metathesis and their biochemical evaluation as lipid II binders: toward the design of potential novel antibiotics.

This report describes the design, synthesis, and biochemical evaluation of alkene- and alkane-bridged AB(C)-ring mimics of the lantibiotic nisin. Nisin belongs to a class of natural antimicrobial peptides, and has a unique mode of action: its AB(C)-ring system binds to the pyrophosphate moiety of lipid II. This mode of action was the rationale for the design of smaller nisin-derived peptides to obtain novel potential antibiotics. As a conformational constraint the thioether bridge was mimicked by an alkene- or alkane isostere. The peptides of the linear individual ring precursors were synthesized on solid support or in solution, and cyclized by ring-closing metathesis in solution with overall yields of between 36 and 89 %. The individual alkene-bridged macrocycles were assembled in solution by using carbodiimide-based synthesis protocols for the corresponding AB(C)-ring mimics. These compounds were tested for their binding affinity toward lipid II by evaluation of their potency to inhibit nisin-induced carboxyfluorescein release from large unilamellar vesicles. It was found that these AB(C)-ring mimics were not able to induce membrane leakage; however, they acted by inhibiting nisin-induced carboxyfluorescein release; this indicates their affinity toward lipid II. These results imply that an alkene or alkane moiety is a suitable thioether bridge mimic.

Step-wise and pre-organization induced synthesis of a crossed alkene-bridged nisin Z DE-ring mimic by ring-closing metathesis.

This paper describes two approaches for the synthesis of a crossed alkene-bridged mimic of the thioether ring system of the nisin Z DE-fragment. The first approach comprised the stepwise total synthesis featuring a cross metathesis and a macrolactamization on a solid support followed by a ring-closing metathesis in solution. Via this route the title compound was obtained in an overall yield of 7% (85% on average for 16 reaction steps). In the second approach, the linear precursor peptide was subjected to ring-closing metathesis and the bicyclic peptide with the correct side chain connectivity pattern was obtained in yields up to 95%. The preferred formation of the bicyclic crossed alkene-bridged mimic of the DE-ring suggests a favorable pre-organization of the linear precursor peptide.

Ring-closing alkyne metathesis approach toward the synthesis of alkyne mimics of thioether A-, B-, C-, and DE-ring systems of the lantibiotic nisin Z.

[reaction: see text] Ring-closing alkyne metathesis toward the synthesis of the alkyne-brigded A-, B-, C-, and (D)E-ring mimics of the peptide antibiotic nisin Z is described. We have successfully synthesized alkyne-bridged cyclic peptides containing 4-7 amino acid residues in yields ranging from 18 to 82%.

PEGylation of the antimicrobial peptide nisin A: problems and perspectives.

Nisin is a natural antimicrobial peptide produced by Lactococcus lactis and widely employed as food preservative. Its low solubility in neutral aqueous solutions, its instability at physiological pH and its rapid breakdown by proteolytic enzymes has limited its use for processed foods (processed cheese, milk and derivatives, canned vegetables). The conjugation to poly(ethylene glycol) (PEG) could improve its solubility and protect it towards enzymes present in non optimally processed food. We report the synthesis of a PEG-nisin conjugate, and the microbiology assays against some bacterial cell lines.

Incorporation of nisin in micro-particles of calcium alginate.

Nisin was successfully incorporated into a matrix of calcium alginate and ground into micro-particles smaller than 150 microns. Formation of micro-particles and incorporation of nisin was verified by scanning electron microscopy and by the reduction in the inactivation of nisin activity with proteolytic enzymes. Incorporation efficiency was 87-93% and the nisin in the alginate-incorporated form was 100% active against an indicator culture of Lactobacillus curvatus both in MRS broth and reconstituted skim milk.