High Charge Density in Peptide Dendrimers is Required to Destabilize Membranes: Insights into Endosome Evasion.

Peptide dendrimers are a type of branched, symmetric, and topologically well-defined molecule that have already been used as delivery systems for nucleic acid transfection. Several of the most promising sequences showed high efficiency in many key steps of transfection, namely, binding siRNA, entering cells, and evading the endosome. However, small changes to the peptide dendrimers, such as in the hydrophobic core, the amino acid chirality, or the total available charges, led to significantly different experimental results with unclear mechanistic insights. In this work, we built a computational model of several of those peptide dendrimers (MH18, MH13, and MH47) and some of their variants to study the molecular details of the structure and function of these molecules. We performed CpHMD simulations in the aqueous phase and in interaction with a lipid bilayer to assess how conformation and protonation are affected by pH in different environments. We found that while the different peptide dendrimer sequences lead to no substantial structural differences in the aqueous phase, the total charge and, more importantly, the total charge density are key for the capacity of the dendrimer to interact and destabilize the membrane. These dendrimers become highly charged when the pH changes from 7.5 to 4.5, and the presence of a high charge density, which is decreased for MH47 that has four fewer titratable lysines, is essential to trigger membrane destabilization. These findings are in excellent agreement with the experimental data and help us to understand the high efficiency of some dendrimers and why the dendrimer MH47 is unable to complete the transfection process. This evidence provides further understanding of the mode of action of these peptide dendrimers and will be pivotal for the future design of new sequences with improved transfection capabilities.

Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase.

C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C-mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound. The structures indicate how the C-mannosylation sequon is recognized by this CMT and its paralogs, and how sequon binding triggers conformational activation of the donor substrate: a process relevant to all glycosyltransferase C superfamily enzymes. Our structural data further indicate that the CMTs adopt an unprecedented electrophilic aromatic substitution mechanism to enable the C-glycosylation of proteins. These results afford opportunities for understanding human disease and therapeutic targeting of specific CMT paralogs.

Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms.

Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits.

Lipophilic Peptide Dendrimers for Delivery of Splice-Switching Oligonucleotides.

Non-viral transfection reagents are continuously being developed in attempt to replace viral vectors. Among those non-viral vectors, dendrimers have gained increasing interest due to their unique molecular structure and multivalency. However, more improvements are still needed to achieve higher efficacy and lower toxicity. In this study, we have examined 18 peptide dendrimers conjugated to lipophilic moieties, such as fatty acids or hydrophobic amino acids, that were previously explored for siRNA. Reporter cells were employed to investigate the transfection of single strand splice-switching oligonucleotides (ONs) using these peptide dendrimers. Luciferase level changes reflecting efficiency varied with amino acid composition, stereochemistry, and complexation media used. 3rd generation peptide dendrimers with D-amino acid configuration were superior to L-form. Lead formulations with 3rd generation, D-amino acid peptide dendrimers increased the correction level of the delivered ON up to 93-fold over untreated HeLa Luc/705 cells with minimal toxicity. To stabilize the formed complexes, Polyvinyl alcohol 18 (PVA18) polymer was added. Although PVA18 addition increased activity, toxicity when using our best candidates G 2,3KL-(Leu)4 (D) and G 2,3KL-diPalmitamide (D) was observed. Our findings demonstrate the potential of lipid-conjugated, D-amino acid-containing peptide dendrimers to be utilized as an effective and safe delivery vector for splice-switching ONs.

A Gold Nanoparticle Nanonuclease Relying on a Zn(II) Mononuclear Complex.

Similarly to enzymes, functionalized gold nanoparticles efficiently catalyze chemical reactions, hence the term nanozymes. Herein, we present our results showing how surface-passivated gold nanoparticles behave as synthetic nanonucleases, able to cleave pBR322 plasmid DNA with the highest efficiency reported so far for catalysts based on a single metal ion mechanism. Experimental and computational data indicate that we have been successful in creating a catalytic site precisely mimicking that suggested for natural metallonucleases relying on a single metal ion for their activity. It comprises one Zn(II) ion to which a phosphate diester of DNA is coordinated. Importantly, as in nucleic acids-processing enzymes, a positively charged arginine plays a key role by assisting with transition state stabilization and by reducing the pKa of the nucleophilic alcohol of a serine. Our results also show how designing a catalyst for a model substrate (bis-p-nitrophenylphosphate) may provide wrong indications as for its efficiency when it is tested against the real target (plasmid DNA).

Transfecting tissue models with CRISPR/Cas9 plasmid DNA using peptide dendrimers.

There is currently a lack of efficient reagents to transfect cells with large plasmid DNA, which would be enabling tools for gene editing using CRISPR/Cas9 technology. Herein, we report the discovery of peptide dendrimer Z22 as a non-viral vector for transfecting large CRISPR/Cas9 pDNA into 3D-tumor spheroids with exceptionally high efficiency, low cytotoxicity and low immunogenicity.

Structure and mechanism of the ER-based glucosyltransferase ALG6.

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.

Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations.

Non-viral transfection vectors are commonly used for oligonucleotide (ON) delivery but face many challenges before reaching the desired compartments inside cells. With the support of additional compounds, it might be more feasible for a vector to endure the barriers and achieve efficient delivery. In this report, we screened 18 different excipients and evaluated their effect on the performance of peptide dendrimer/lipid vector to deliver single-stranded, splice-switching ONs under serum conditions. Transfection efficiency was monitored in four different reporter cell lines by measuring splice-switching activity on RNA and protein levels. All reporter cell lines used had a mutated human ß-globin intron 2 sequence interrupting the luciferase gene, which led to an aberrant splicing of luciferase pre-mRNA and subsidence of luciferase protein translation. In the HeLa Luc/705 reporter cell line (a cervical cancer cell line), the lead excipients (Polyvinyl derivatives) potentiated the splice-switching activity up to 95-fold, compared to untreated cells with no detected cytotoxicity. Physical characterization revealed that lead excipients decreased the particle size and the zeta potential of the formulations. In vivo biodistribution studies emphasized the influence of formulations as well as the type of excipients on biodistribution profiles of the ON. Subsequently, we suggest that the highlighted impact of tested excipients would potentially assist in formulation development to deliver ON therapeutics in pre-clinical and clinical settings.

Fluorescence Imaging of Bacterial Killing by Antimicrobial Peptide Dendrimer G3KL.

We recently discovered that peptide dendrimers such as G3KL ((KL)8(KKL)4(KKL)2KKL, K = branching l-lysine) exert strong activity against Gram-negative bacteria including Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. Herein, we report a detailed mechanistic study using fluorescence labeled analogs bearing fluorescein (G3KL-Fluo) or dansyl (G3KL-Dansyl), which show a similar bioactivity profile as G3KL. Imaging bacterial killing by super-resolution stimulated emission depletion (STED) microscopy, time-lapse imaging, and transmission electron microscopy (TEM) reveals that the dendrimer localizes at the bacterial membrane, induces membrane depolarization and permeabilization, and destroys the outer leaflet and the inner membrane. G3KL accumulates in bacteria against which it is active; however, it only weakly penetrates into eukaryotic cells without inducing significant toxicity. G3KL furthermore binds to lipopolysaccharide (LPS) and inhibits the LPS induced release of TNF-α by macrophages, similarly to polymyxin B. Taken together, these experiments show that G3KL behaves as a potent membrane disruptive antimicrobial peptide.

Glycocluster Tetrahydroxamic Acids Exhibiting Unprecedented Inhibition of Pseudomonas aeruginosa Biofilms.

Opportunistic Gram-negative Pseudomonas aeruginosa uses adhesins (e.g., LecA and LecB lectins, type VI pili and flagella) and iron to invade host cells with the formation of a biofilm, a thick barrier that protects bacteria from drugs and host immune system. Hindering iron uptake and disrupting adhesins' function could be a relevant antipseudomonal strategy. To test this hypothesis, we designed an iron-chelating glycocluster incorporating a tetrahydroxamic acid and α-l-fucose bearing linker to interfere with both iron uptake and the glycan recognition process involving the LecB lectin. Iron depletion led to increased production of the siderophore pyoverdine by P. aeruginosa to counteract the loss of iron uptake, and strong biofilm inhibition was observed not only with the α-l-fucocluster (72%), but also with its α-d-manno (84%), and α-d-gluco (92%) counterparts used as negative controls. This unprecedented finding suggests that both LecB and biofilm inhibition are closely related to the presence of hydroxamic acid groups.

Stereoselective pH Responsive Peptide Dendrimers for siRNA Transfection.

Transfecting nucleic acids into cells is an essential procedure in biological research usually performed using nonviral transfection reagents. Unfortunately, most transfection reagents have polymeric or undisclosed structures and require nonstandard synthetic procedures. Herein we report peptide dendrimers accessible as pure products from standard building blocks by solid-phase peptide synthesis and acting as nontoxic single component siRNA transfection reagents for a variety of cell lines with equal or better performance than the gold standard lipofectamine L2000. Structure-activity relationships and mechanistic studies illuminate their transfection mechanism in unprecedented detail. Stereoselective dendrimer aggregation via intermolecular ß-sheets at neutral pH enables siRNA complexation to form nanoparticles which enter cells by endocytosis. Endosome acidification triggers protonation of amino termini and rearrangement to an α-helical conformation forming smaller dendrimer/siRNA nanoparticles, which escape the endosome and release their siRNA cargo in the cytosol. Two particularly efficient d-enantiomeric dendrimers are proposed as new reference reagents for siRNA transfection.

Factors Influencing the Activity of Nanozymes in the Cleavage of an RNA Model Substrate.

A series of 2-nm gold nanoparticles passivated with different thiols all featuring at least one triazacyclonanone-Zn(II) complex and different flanking units (a second Zn(II) complex, a triethyleneoxymethyl derivative or a guanidinium of arginine of a peptide) were prepared and studied for their efficiency in the cleavage of the RNA-model substrate 2-hydroxypropyl-p-nitrophenyl phosphate. The source of catalysis for each of them was elucidated from the kinetic analysis (Michaelis-Menten profiles, pH dependence and kinetic isotope effect). The data indicated that two different mechanisms were operative: One involving two Zn(II) complexes and the other one involving a single Zn(II) complex and a flanking guanidinium cation. The mechanism based on a dinuclear catalytic site appeared more efficient than the one based on the cooperativity between a metal complex and a guanidinium.

X-ray Crystal Structures of Short Antimicrobial Peptides as Pseudomonas aeruginosa Lectin B Complexes.

Herein, we report X-ray crystal structures of 11-13 residue antimicrobial peptides (AMPs) active against Pseudomonas aeruginosa as complexes of fucosylated d-enantiomeric sequences with the P. aeruginosa lectin LecB. These represent the first crystal structures of short AMPs. In 24 individual structures of eight different peptides, we found mostly α-helices assembled as two-helix or four-helix bundles with a hydrophobic core and cationic residues pointing outside. Two of the analogs formed an extended structure engaging in multiple contacts with the lectin. Molecular dynamics (MD) simulations showed that α-helices are stabilized by bundle formation and suggested that the N-terminal acyl group present in the linker to the fucosyl group can extend the helix by one additional H-bond and increase α-helix amphiphilicity. Investigating N-terminal acylation led to AMPs with equivalent and partly stronger antibacterial effects compared to the free peptide.

Structure of bacterial oligosaccharyltransferase PglB bound to a reactive LLO and an inhibitory peptide.

Oligosaccharyltransferase (OST) is a key enzyme of the N-glycosylation pathway, where it catalyzes the transfer of a glycan from a lipid-linked oligosaccharide (LLO) to an acceptor asparagine within the conserved sequon N-X-T/S. A previous structure of a ternary complex of bacterial single subunit OST, PglB, bound to a non-hydrolyzable LLO analog and a wild type acceptor peptide showed how both substrates bind and how an external loop (EL5) of the enzyme provided specific substrate-binding contacts. However, there was a relatively large separation of the substrates at the active site. Here we present the X-ray structure of PglB bound to a reactive LLO analog and an inhibitory peptide, revealing previously unobserved interactions in the active site. We found that the atoms forming the N-glycosidic bond (C-1 of the GlcNAc moiety of LLO and the -NH2 group of the peptide) are closer than in the previous structure, suggesting that we have captured a conformation closer to the transition state of the reaction. We find that the distance between the divalent metal ion and the glycosidic oxygen of LLO is now 4 Å, suggesting that the metal stabilizes the leaving group of the nucleophilic substitution reaction. Further, the carboxylate group of a conserved aspartate of PglB mediates an interaction network between the reducing-end sugar of the LLO, the asparagine side chain of the acceptor peptide, and a bound divalent metal ion. The interactions identified in this novel state are likely to be relevant in the catalytic mechanisms of all OSTs.

Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity.

Despite the advances in gene therapy and in oligonucleotide (ON) chemistry, efficient cellular delivery remains an obstacle. Most current transfection reagents suffer from low efficacy or high cytotoxicity. In this report, we describe the synergism between lipid and dendrimer delivery vectors to enhance the transfection efficiency, while avoiding high toxicity. We screened a library of 20 peptide dendrimers representing three different generations and evaluated their capability to deliver a single-stranded splice-switching ON after formulating with lipids (DOTMA/DOPE). The transfection efficiency was analyzed in 5 reporter cell lines, in serum-free and serum conditions, and with 5 different formulation protocols. All formulations displayed low cytotoxicity to the majority of the tested cell lines. The complex sizes were < 200 nm; particle size distributions of effective mixtures were < 80 nm; and, the zeta potential was dependent on the formulation buffer used. The best dendrimer enhanced transfection in a HeLa reporter cell line by 30-fold compared to untreated cells under serum-free conditions. Interestingly, addition of sucrose to the formulation enabled - for the first time - peptide dendrimers/lipid complexes to efficiently deliver splice-switching ON in the presence of serum, reaching 40-fold increase in splice switching. Finally, in vivo studies highlighted the potential of these formulae to change the biodistribution pattern to be more towards the liver (90% of injected dose) compared to the kidneys (5% of injected dose) or to unformulated ON. This success encourages further development of peptide dendrimer complexes active in serum and future investigation of mechanisms behind the influence of additives on transfection efficacy.

Structural basis of the molecular ruler mechanism of a bacterial glycosyltransferase.

The membrane-associated, processive and retaining glycosyltransferase PglH from Campylobacter jejuni is part of the biosynthetic pathway of the lipid-linked oligosaccharide (LLO) that serves as the glycan donor in bacterial protein N-glycosylation. Using an unknown counting mechanism, PglH catalyzes the transfer of exactly three α1,4 N-acetylgalactosamine (GalNAc) units to the growing LLO precursor, GalNAc-α1,4-GalNAc-α1,3-Bac-α1-PP-undecaprenyl. Here, we present crystal structures of PglH in three distinct states, including a binary complex with UDP-GalNAc and two ternary complexes containing a chemo-enzymatically generated LLO analog and either UDP or synthetic, nonhydrolyzable UDP-CH2-GalNAc. PglH contains an amphipathic helix ("ruler helix") that has a dual role of facilitating membrane attachment and glycan counting. The ruler helix contains three positively charged side chains that can bind the pyrophosphate group of the LLO substrate and thus limit the addition of GalNAc units to three. These results, combined with molecular dynamics simulations, provide the mechanism of glycan counting by PglH.

Lipidated Peptide Dendrimers Killing Multidrug-Resistant Bacteria.

New antibiotics are urgently needed to address multidrug-resistant (MDR) bacteria. Herein we report that second-generation (G2) peptide dendrimers bearing a fatty acid chain at the dendrimer core efficiently kill Gram-negative bacteria including Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most problematic MDR bacteria worldwide. Our most active dendrimer TNS18 is also active against Gram-positive methicillin-resistant Staphylococcus aureus. Based on circular dichroism and molecular dynamics studies, we hypothesize that TNS18 adopts a hydrophobically collapsed conformation in water with the fatty acid chain backfolded onto the peptide dendrimer branches and that the dendrimer unfolds in contact with the membrane to expose its lipid chain and hydrophobic residues, thereby facilitating membrane disruption leading to rapid bacterial cell death. Dendrimer TNS18 shows promising in vivo activity against MDR clinical isolates of A. baumannii and Escherichia coli, suggesting that lipidated peptide dendrimers might become a new class of antibacterial agents.

Chemical space guided discovery of antimicrobial bridged bicyclic peptides against Pseudomonas aeruginosa and its biofilms.

Herein we report the discovery of antimicrobial bridged bicyclic peptides (AMBPs) active against Pseudomonas aeruginosa, a highly problematic Gram negative bacterium in the hospital environment. Two of these AMBPs show strong biofilm inhibition and dispersal activity and enhance the activity of polymyxin, currently a last resort antibiotic against which resistance is emerging. To discover our AMBPs we used the concept of chemical space, which is well known in the area of small molecule drug discovery, to define a small number of test compounds for synthesis and experimental evaluation. Our chemical space was calculated using 2DP, a new topological shape and pharmacophore fingerprint for peptides. This method provides a general strategy to search for bioactive peptides with unusual topologies and expand the structural diversity of peptide-based drugs.

Design, crystal structure and atomic force microscopy study of thioether ligated d,l-cyclic antimicrobial peptides against multidrug resistant Pseudomonas aeruginosa.

Here we report a new family of cyclic antimicrobial peptides (CAMPs) targeting MDR strains of Pseudomonas aeruginosa. These CAMPs are cyclized via a xylene double thioether bridge connecting two cysteines placed at the ends of a linear amphiphilic alternating d,l-sequence composed of lysines and tryptophans. Investigations by transmission electron microscopy (TEM), dynamic light scattering and atomic force microscopy (AFM) suggest that these peptide macrocycles interact with the membrane to form lipid-peptide aggregates. Amphiphilic conformations compatible with membrane disruption are observed in high resolution X-ray crystal structures of fucosylated derivatives in complex with lectin LecB. The potential for optimization is highlighted by N-methylation of backbone amides leading to derivatives with similar antimicrobial activity but lower hemolysis.

Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase.

Oligosaccharyltransferase (OST) is a membrane-integral enzyme that catalyzes the transfer of glycans from lipid-linked oligosaccharides (LLOs) onto asparagine side chains, the first step in protein N-glycosylation. Here, we report the X-ray structure of a single-subunit OST, PglB from Campylobacter lari, trapped in an intermediate state bound to an acceptor peptide and a synthetic LLO analog. The structure reveals the role of the external loop EL5, present in all OST enzymes, in substrate recognition. Whereas the N-terminal half of EL5 binds LLO, the C-terminal half interacts with the acceptor peptide. The glycan moiety of LLO must thread under EL5 to access the active site. Reducing EL5 mobility decreases the catalytic rate of OST when full-size heptasaccharide LLO is provided, but not for a monosaccharide-containing LLO analog. Our results define the chemistry of a ternary complex state, assign functional roles to conserved OST motifs, and provide opportunities for glycoengineering by rational design of PglB.