Structural Determinants in the Staphylococcus aureus-Derived Phenol-Soluble Modulin α2 Peptide Required for Neutrophil Formyl Peptide Receptor Activation.

Highly pathogenic Staphylococcus aureus strains produce phenol-soluble modulins (PSMs), which are N-formylated peptides. Nanomolar concentrations of PSMα2 are recognized by formyl peptide receptor 2 (FPR2), but unlike the prototypic FPR2 agonist WKYMVM, PSMα2 is a biased signaling agonist. The truncated N-terminal PSMα2 variant, consisting of the five N-terminal residues, is no longer recognized by FPR2, showing that the C-terminal part of PSMα2 confers FPR2 selectivity, whereas the N-terminal part may interact with the FPR1 binding site. In the current study, a combined pharmacological and genetic approach involving primary human neutrophils and engineered FPR knock-in and knockout cells was used to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides as well as the receptor downstream signaling induced by these peptides. In comparison with the full-length PSMα2, we show that the peptide in which the N-terminal part of PSMα2 was replaced by fMet-Ile-Phe-Leu (an FPR1-selective peptide agonist) potently activates both FPRs for production of superoxide anions and ß-arrestin recruitment. A shortened analog of PSMα2 (PSMα21-12), lacking the nine C-terminal residues, activated both FPR1 and FPR2 to produce reactive oxygen species, whereas ß-arrestin recruitment was only mediated through FPR1. However, a single amino acid replacement (Gly-2 to Ile-2) in PSMα21-12 was sufficient to alter FPR2 signaling to include ß-arrestin recruitment, highlighting a key role of Gly-2 in conferring FPR2-biased signaling. In conclusion, we provide structural insights into FPR1 and FPR2 recognition as well as the signaling induced by interaction with formyl peptides derived from PSMα2, originating from S. aureus bacteria.

Inoculum effect of antimicrobial peptides.

The activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 105 cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide-cell association, and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered.

Physiological response in E. coli to YdgR overexpression depends on whether the protein has an intact function.

Membrane transport proteins are essential for the transport of a wide variety of molecules across the cell membrane to maintain cellular homeostasis. Generally, these transport proteins can be overexpressed in a suitable host (bacteria, yeast, or mammalian cells), and it is well documented that overexpression of membrane proteins alters the global metabolomic and proteomic profiles of the host cells. In the present study, we investigated the physiological consequences of overexpression of a membrane transport protein YdgR that belongs to the POT/PTR family from E. coli by using the lab strain BL21 (DE3)pLysS in its functional and attenuated mutant YdgR-E33Q. We found significant differences between the omics (metabolomics and proteomics) profiles of the cells expressing functional YdgR as compared to cells expressing attenuated YdgR, e.g., upregulation of several uncharacterized y-proteins and enzymes involved in the metabolism of peptides and amino acids. Furthermore, molecular network analysis suggested a relatively higher presence of proline-containing tripeptides in cells expressing functional YdgR. We envisage that an in-depth investigation of physiological alterations due to protein over-expression may be used for the deorphanization of the y-gene transportome.

The Y-ome Conundrum: Insights into Uncharacterized Genes and Approaches for Functional Annotation.

The ever-increasing availability of genome sequencing data has revealed a substantial number of uncharacterized genes without known functions across various organisms. The first comprehensive genome sequencing of E. coli K12 revealed that more than 50% of its open reading frames corresponded to transcripts with no known functions. The group of protein-coding genes without a functional description and/or a recognized pathway, beginning with the letter "Y", is classified as the "y-ome". Several efforts have been made to elucidate the functions of these genes and to recognize their role in biological processes. This review provides a brief update on various strategies employed when studying the y-ome, such as high-throughput experimental approaches, comparative omics, metabolic engineering, gene expression analysis, and data integration techniques. Additionally, we highlight recent advancements in functional annotation methods, including the use of machine learning, network analysis, and functional genomics approaches. Novel approaches are required to produce more precise functional annotations across the genome to reduce the number of genes with unknown functions.

Structure-activity study of oncocin: On-resin guanidinylation and incorporation of homoarginine, 4-hydroxyproline or 4,4-difluoroproline residues.

Antimicrobial peptides (AMPs) often display guanidinium functionalities, and hence robust synthetic procedures are needed to facilitate access to analogues with unnatural homologues of arginine (Arg = R). Initially, a resin-bound Arg/Pro-rich fluoren-9-yl-methyloxycarbonyl-protected fragment (Fmoc-RPRPPR) of the AMP oncocin (i.e., VDKPPYLPRPRPPRRIYNR-NH2) was employed in a comparative on-resin assessment of commercial guanidinylation reagents head-to-head with the recently studied bis-Boc-protected triazole-based reagent, 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]-carboxamidine, which was synthesized by a chromatography-free procedure. This reagent was found to enable quantitative conversion in solid-phase peptide synthesis (SPPS) of peptides displaying homoarginine (Har) residues and/or an N-terminal guanidinium group. SPPS was used to obtain analogues of the 18-mer oncocin with single as well as multiple Arg → Har modifications. In addition, the effect of replacement of proline (Pro) residues in oncocin was explored by incorporating single or multiple trans-4-hydroxy-l-proline (Hyp) or 4,4-difluoro-l-proline (Dfp) residues, which both affected hydrophobicity. The resulting peptide library was tested against both Gram-negative and Gram-positive bacteria. Analysis of the minimal inhibitory concentrations (MICs) showed that analogues, displaying modifications at positions 4, 5 and 12 (originally Pro residues), had retained or slightly improved antimicrobial activity. Next, an oncocin analogue with two stabilizing l-Arg → d-Arg replacements in the C-terminal part was further modified by triple-replacement of Pro by either Dfp or Hyp in positions 4, 5, and 12. The resulting analogue displaying three Pro → Dfp modifications proved to possess the best activity profile: MICs of 1-2 µg/mL against E. coli and Klebsiella pneumoniae, less than 1% hemolysis at 800 µg/mL, and an IC50 above 1280 µg/mL in HepG2 cells. Thus, incorporation of bis-fluorinated Pro residues appears to constitute a novel tool in structure-activity studies aimed at optimization of Pro-rich AMPs.

Selected strategies to fight pathogenic bacteria.

Natural products and analogues are a source of antibacterial drug discovery. Considering drug resistance levels emerging for antibiotics, identification of bacterial metalloenzymes and the synthesis of selective inhibitors are interesting for antibacterial agent development. Peptide nucleic acids are attractive antisense and antigene agents representing a novel strategy to target pathogens due to their unique mechanism of action. Antisense inhibition and development of antisense peptide nucleic acids is a new approach to antibacterial agents. Due to the increased resistance of biofilms to antibiotics, alternative therapeutic options are necessary. To develop antimicrobial strategies, optimised in vitro and in vivo models are needed. In vivo models to study biofilm-related respiratory infections, device-related infections: ventilator-associated pneumonia, tissue-related infections: chronic infection models based on alginate or agar beads, methods to battle biofilm-related infections are discussed. Drug delivery in case of antibacterials often is a serious issue therefore this review includes overview of drug delivery nanosystems.

Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity.

In vitro determination of hemolytic properties is a common and important method for preliminary evaluation of cytotoxicity of chemicals, drugs, or any blood-contacting medical device or material. The method itself is relatively straightforward, however, protocols used in the literature vary substantially. This leads to significant difficulties both in interpreting and in comparing the obtained values. Here, we examine how the different variables used under different experimental setups may affect the outcome of this assay. We find that certain key parameters affect the hemolysis measurements in a critical manner. The hemolytic effect of compounds tested here varied up to fourfold depending on the species of the blood source. The use of different types of detergents used for generating positive control samples (i.e., 100% hemolysis) produced up to 2.7-fold differences in the calculated hemolysis ratios. Furthermore, we find an expected, but substantial, increase in the number of hemolyzed erythrocytes with increasing erythrocyte concentration and with prolonged incubation time, which in turn affects the calculated hemolysis ratios. Based on our findings we propose an optimized protocol in an attempt to standardize future hemolysis studies.

Perfluoro-tert-butanol for selective on-resin detritylation: a mild alternative to traditionally used methods.

Solid-phase synthesis of cyclic, branched or side-chain-modified peptides typically involves introduction of a residue carrying a temporary side-chain protecting group that undergoes selective on-resin removal. In particular, Nα-Fmoc-Nε-(4-methyltriphenylmethyl) (Mtt)-protected lysine and its shorter analogues are commercially available and extensively used in this context. Nevertheless, rapid reliable methods for on-resin removal of Mtt groups in the presence of tert-butyloxycarbonyl (Boc) groups are needed. Current commonly used conditions involve low concentrations (1-3%) of trifluoroacetic acid (TFA) in dichloromethane, albeit adjustment to each specific application is required to avoid premature removal of Boc groups or cleavage from the linker. Hence, a head-to-head comparison of several deprotection conditions was performed. The selected acids represent a wide range of acidity from TFA to trifluoroethanol. Also, on-resin removal of the N-(4-methoxytriphenylmethyl) (Mmt) and O-trityl groups (on serine) was investigated under similar conditions. The mildest conditions identified for Mtt deprotection involve successive treatments with 30% hexafluoroisopropanol (HFIP) or 30% perfluoro-tert-butanol [(CF3)3COH] in dichloromethane (3 × 5 or 3 × 15 min, respectively), while 30% HFIP, 30% (CF3)3COH, or 10% AcOH-20% trifluoroethanol (TFE) in CH2Cl2 (3 × 5 min) as well as 5% trichloroacetic acid in CH2Cl2 (3 × 2 min) enabled Mmt removal. Treatment with 1% TFA with/without 2% triisopropylsilane added (3 × 5 min), but also prolonged treatment with 30% (CF3)3COH (5 × 15 min), led to selective deprotection of an O-Trt group on a serine residue. In all cases, the sequences also contained N-Boc or O-tBu protecting groups, which were not affected by 30% HFIP or 30% (CF3)3COH even after a prolonged reaction time of 4 h. Finally, the optimized conditions involving HFIP or (CF3)3COH proved applicable also for selective deprotection of a longer resin-bound peptide [i.e., Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile-Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] as well as allowed for an almost complete deprotection of a Dab(Mtt) residue.

Evaluation of 1H-Triazole-1-[N,N'-Bis(tert-butoxycarbonyl)]carboxamidine in Solution-Phase and On-Resin Guanidinylation.

Several guanidines and guanidinylated peptides have substantial potential as therapeutics, but efficient guanidinylation reagents are vital for easy access to these compounds. Presently, pyrazole-1-carboxamidine type reagents are commonly used in the transformations of amines into corresponding guanidines. Here, we report a comparative study of the utility of 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine, which was synthesized in two steps and readily upscaled to gram amounts. It exhibited excellent performance in solution-phase reactions, rapidly converting a set of representative aliphatic primary and unhindered secondary amines as well as aniline into the corresponding bis(tert-butoxycarbonyl)-protected guanidines. To enable a direct assessment of the reactivity of guanidinylation reagents, conversions were performed in deuterated solvents (d7-DMF or d8-THF), allowing for continuous analysis of the reaction mixtures by 1H and 13C NMR. Likewise, 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine proved to be a versatile reagent in solid-phase conversions, for example, a resin-bound test peptide (KFFKFFK) was fully guanidinylated in only 2 h by using 2 equivalents of the reagent per free amino group. Also, 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine proved capable of completely guanidinylating more sterically hindered N-terminal residues (e.g., N-methyl amino acids or a peptoid) in resin-bound peptides. Its superior reactivity and stability demonstrated under heating conditions make 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine a valuable guanidinylation reagent both in solution- and solid-phase synthesis.

Macrophage Phosphoproteome Analysis Reveals MINCLE-dependent and -independent Mycobacterial Cord Factor Signaling.

Immune sensing of Mycobacterium tuberculosis relies on recognition by macrophages. Mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM), is the most abundant cell wall glycolipid and binds to the C-type lectin receptor (CLR) MINCLE. To explore the kinase signaling linking the TDM-MINCLE interaction to gene expression, we employed quantitative phosphoproteome analysis. TDM caused upregulation of 6.7% and suppressed 3.8% of the 14,000 phospho-sites identified on 3727 proteins. MINCLE-dependent phosphorylation was observed for canonical players of CLR signaling (e.g. PLCγ, PKCδ), and was enriched for PKCδ and GSK3 kinase motifs. MINCLE-dependent activation of the PI3K-AKT-GSK3 pathway contributed to inflammatory gene expression and required the PI3K regulatory subunit p85α. Unexpectedly, a substantial fraction of TDM-induced phosphorylation was MINCLE-independent, a finding paralleled by transcriptome data. Bioinformatics analysis of both data sets concurred in the requirement for MINCLE for innate immune response pathways and processes. In contrast, MINCLE-independent phosphorylation and transcriptome responses were linked to cell cycle regulation. Collectively, our global analyses show substantial reprogramming of macrophages by TDM and reveal a dichotomy of MINCLE-dependent and -independent signaling linked to distinct biological responses.

Peptide/ß-Peptoid Hybrids with Activity against Vancomycin-Resistant Enterococci: Influence of Hydrophobicity and Structural Features on Antibacterial and Hemolytic Properties.

Infections with enterococci are challenging to treat due to intrinsic resistance to several antibiotics. Especially vancomycin-resistant Enterococcus faecium and Enterococcus faecalis are of considerable concern with a limited number of efficacious therapeutics available. From an initial screening of 20 peptidomimetics, 11 stable peptide/ß-peptoid hybrids were found to have antibacterial activity against eight E. faecium and E. faecalis isolates. Microbiological characterization comprised determination of minimal inhibitory concentrations (MICs), probing of synergy with antibiotics in a checkerboard assay, time-kill studies, as well as assessment of membrane integrity. E. faecium isolates proved more susceptible than E. faecalis isolates, and no differences in susceptibility between the vancomycin-resistant (VRE) and -susceptible E. faecium isolates were observed. A test of three peptidomimetics (Ac-[hArg-ßNsce]6-NH2, Ac-[hArg-ßNsce-Lys-ßNspe]3-NH2 and Oct-[Lys-ßNspe]6-NH2) in combination with conventional antibiotics (vancomycin, gentamicin, ciprofloxacin, linezolid, rifampicin or azithromycin) revealed no synergy. The same three potent analogues were found to have a bactericidal effect with a membrane-disruptive mode of action. Peptidomimetics Ac-[hArg-ßNsce-Lys-ßNspe]3-NH2 and Oct-[Lys-ßNspe]6-NH2 with low MIC values (in the ranges 2-8 µg/mL and 4-16 µg/mL against E. faecium and E. faecalis, respectively) and displaying weak cytotoxic properties (i.e., <10% hemolysis at a ~100-fold higher concentration than their MICs; IC50 values of 73 and 41 µg/mL, respectively, against HepG2 cells) were identified as promising starting points for further optimization studies.

Peptide/ß-Peptoid Hybrids with Ultrashort PEG-Like Moieties: Effects on Hydrophobicity, Antibacterial Activity and Hemolytic Properties.

PEGylation of antimicrobial peptides as a shielding tool that increases stability toward proteolytic degradation typically leads to concomitant loss of activity, whereas incorporation of ultrashort PEG-like amino acids (sPEGs) remains essentially unexplored. Here, modification of a peptide/ß-peptoid hybrid with sPEGs was examined with respect to influence on hydrophobicity, antibacterial activity and effect on viability of mammalian cells for a set of 18 oligomers. Intriguingly, the degree of sPEG modification did not significantly affect hydrophobicity as measured by retention in reverse-phase HPLC. Antibacterial activity against both wild-type and drug-resistant strains of Escherichia coli and Acinetobacter baumannii (both Gram-negative pathogens) was retained or slightly improved (MICs in the range 2-16 µg/mL equal to 0.7-5.2 µM). All compounds in the series exhibited less than 10% hemolysis at 400 µg/mL. While the number of sPEG moieties appeared not to be clearly correlated with hemolytic activity, a trend toward slightly increased hemolytic activity was observed for analogues displaying the longest sPEGs. In contrast, within a subseries the viability of HepG2 liver cells was least affected by analogues displaying the longer sPEGs (with IC50 values of ~1280 µg/mL) as compared to most other analogues and the parent peptidomimetic (IC50 values in the range 330-800 µg/mL).

Targeting Toxins toward Tumors.

Many cancer diseases, e.g., prostate cancer and lung cancer, develop very slowly. Common chemotherapeutics like vincristine, vinblastine and taxol target cancer cells in their proliferating states. In slowly developing cancer diseases only a minor part of the malignant cells will be in a proliferative state, and consequently these drugs will exert a concomitant damage on rapidly proliferating benign tissue as well. A number of toxins possess an ability to kill cells in all states independently of whether they are benign or malignant. Such toxins can only be used as chemotherapeutics if they can be targeted selectively against the tumors. Examples of such toxins are mertansine, calicheamicins and thapsigargins, which all kill cells at low micromolar or nanomolar concentrations. Advanced prodrug concepts enabling targeting of these toxins to cancer tissue comprise antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzyme-activated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC), which will be discussed in the present review. The review also includes recent examples of protease-targeting chimera (PROTAC) for knockdown of receptors essential for development of tumors. In addition, targeting of toxins relying on tumor-overexpressed enzymes with unique substrate specificity will be mentioned.

The Effects of Structural Alterations in the Polyamine and Amino Acid Moieties of Philanthotoxins on Nicotinic Acetylcholine Receptor Inhibition in the Locust, Schistocerca gregaria.

Alterations in the polyamine and amino acid (tyrosine) moieties of philanthotoxin-343 (PhTX-343) were investigated for their effects on the antagonism of nicotinic acetylcholine receptors (nAChRs) isolated from the locust (Schistocerca gregaria) mushroom body. Through whole-cell patch-clamp recordings, the philanthotoxin analogues in this study were shown to cause inhibition of the inward current when co-applied with acetylcholine (ACh). PhTX-343 (IC50 = 0.80 µM at -75 mV) antagonised locust nAChRs in a use-dependent manner, suggesting that it acts as an open-channel blocker. The analogue in which both the secondary amine functionalities were replaced with methylene groups (i.e., PhTX-12) was ~6-fold more potent (IC50 (half-maximal inhibitory concentration) = 0.13 µM at -75 mV) than PhTX-343. The analogue containing cyclohexylalanine as a substitute for the tyrosine moiety of PhTX-343 (i.e., Cha-PhTX-343) was also more potent (IC50 = 0.44 µM at -75 mV). A combination of both alterations to PhTX-343 generated the most potent analogue, i.e., Cha-PhTX-12 (IC50 = 1.71 nM at -75 mV). Modulation by PhTX-343 and Cha-PhTX-343 fell into two distinct groups, indicating the presence of two pharmacologically distinct nAChR groups in the locust mushroom body. In the first group, all concentrations of PhTX-343 and Cha-PhTX-343 inhibited responses to ACh. In the second group, application of PhTX-343 or Cha-PhTX-343 at concentrations ≤100 nM caused potentiation, while concentrations ≥ 1 µM inhibited responses to ACh. Cha-PhTX-12 may have potential to be developed into insecticidal compounds with a novel mode of action.

Novel Cyclic Lipopeptide Antibiotics: Effects of Acyl Chain Length and Position.

Multidrug-resistant bacteria are a global health problem. One of the last-resort antibiotics against Gram-negative bacteria is the cyclic lipopeptide colistin, displaying a flexible linker with a fatty acid moiety. The aim of the present project was to investigate the effect on antimicrobial activity of introducing fatty acid moieties of different lengths and in different positions in a cyclic peptide, S3(B), containing a flexible linker. The lipidated analogues of S3(B) were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis. Following assembly of the linear peptide by Fmoc solid-phase peptide synthesis, on-resin head-to-tail cyclization and fatty acid acylation were performed. The antimicrobial activity was determined against the ESKAPE pathogens, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, hemolytic activity was determined against human erythrocytes. A total of 18 cyclic lipopeptides were synthesized and characterized. It was found that introduction of fatty acids in positions next to the flexible linker was more strongly linked to antimicrobial activity. The fatty acid length altered the overall hydrophobicity, which was the driving force for both high antimicrobial and hemolytic activity. Peptides became highly hemolytic when carbon-chain length exceeded 10 (i.e., C10), overlapping with the optimum for antimicrobial activity (i.e., C8-C12). The most promising candidate (C8)5 showed antimicrobial activity corresponding to that of S3(B), but with an improved hemolytic profile. Finally, (C8)5 was further investigated in a time-kill experiment.

Fluorescent Analogues of Human α-Calcitonin Gene-Related Peptide with Potent Vasodilator Activity.

: Human α-calcitonin gene-related peptide (h-α-CGRP) is a highly potent vasodilator peptide that belongs to the family of calcitonin peptides. There are two forms of CGRP receptors in humans and rodents: α-CGRP receptor predominately found in the cardiovascular system and ß-CGRP receptor predominating in the gastrointestinal tract. The CGRP receptors are primarily localized to C and Aδ sensory fibers, where they are involved in nociceptive transmission and migraine pathophysiology. These fibers are found both peripherally and centrally, with extensive perivascular location. The CGRP receptors belong to the class B G-protein-coupled receptors, and they are primarily associated to signaling via Gα proteins. The objectives of the present work were: (i) synthesis of three single-labelled fluorescent analogues of h-α-CGRP by 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis, and (ii) testing of their biological activity in isolated human, mouse, and rat arteries by using a small-vessel myograph setup. The three analogues were labelled with 5(6)-carboxyfluorescein via the spacer 6-aminohexanoic acid at the chain of Lys24 or Lys35. Circular dichroism (CD) experiments were performed to obtain information on the secondary structure of these fluorescently labelled peptides. The CD spectra indicated that the folding of all three analogues was similar to that of native α-CGRP. The three fluorescent analogues of α-CGRP were successfully prepared with a purity of >95%. In comparison to α-CGRP, the three analogues exhibited similar efficacy, but different potency in producing a vasodilator effect. The analogue labelled at the N-terminus proved to be the most readily synthesized, but it was found to possess the lowest vasodilator potency. The analogues labelled at Lys35 or Lys24 exhibited an acceptable reduction in potency (i.e., 3-5 times and 5-10 times less potent, respectively), and thus they have potential for use in further investigations of receptor internalization and neuronal reuptake.

19F-substituted amino acids as an alternative to fluorophore labels: monitoring of degradation and cellular uptake of analogues of penetratin by 19F NMR.

Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of 19F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using 19F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of 19F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.

Studies on acid stability and solid-phase block synthesis of peptide-peptoid hybrids: ligands for formyl peptide receptors.

α-Peptoids as well as peptide/α-peptoid hybrids and peptide/ß-peptoid hybrids constitute major classes of proteolytically stable peptidomimetics that have been extensively investigated as mimetics of biologically active peptides. Representatives of lipidated peptide/ß-peptoid hybrids have been identified as promising immunomodulatory lead compounds, and hence access to these via protocols suitable for gram-scale synthesis is warranted to enable animal in vivo studies. Recent observations indicated that several byproducts appear in crude mixtures of relatively short benzyl-based peptide/ß-peptoid oligomers, and that these were most predominant when the ß-peptoid units displayed an α-chiral benzyl side chain. This prompted an investigation of their stability under acidic conditions. Simultaneous deprotection and cleavage of peptidomimetics containing either α-chiral α- or ß-peptoid residues required treatment with strong acid only for a short time to minimize the formation of partially debenzylated byproducts. The initial work on peptide/ß-peptoid oligomers with an alternating design established that it was beneficial to form the amide bond between the carboxyl group of the α-amino acid and the congested amino functionality of the ß-peptoid residue in solution. To further simplify oligomer assembly on solid phase, we now present a protocol for purification-free solid-phase synthesis of tetrameric building blocks. Next, syntheses of peptidomimetic ligands via manual solid-phase methodologies involving tetrameric building blocks were found to give more readily purified products as compared to those obtained with dimeric building blocks. Moreover, the tetrameric building blocks could be utilized in automated synthesis with microwave-assisted heating, albeit the purity of the crude products was not increased.

Application of a Quality-By-Design Approach to Optimise Lipid-Polymer Hybrid Nanoparticles Loaded with a Splice-Correction Antisense Oligonucleotide: Maximising Loading and Intracellular Delivery.

BACKGROUND: Antisense oligonucleotides (ASOs) are promising therapeutics for specific modulation of cellular RNA function. However, ASO efficacy is compromised by inefficient intracellular delivery. Lipid-polymer hybrid nanoparticles (LPNs) are attractive mediators of intracellular ASO delivery due to favorable colloidal stability and sustained release properties. METHODS: LPNs composed of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) were studied for delivery of an ASO mediating splice correction of a luciferase gene transcript (Luc-ASO). Specific purposes were: (i) to increase the mechanistic understanding of factors determining the loading of ASO in LPNs, and (ii) to optimise the LPNs and customise them for Luc-ASO delivery in HeLa pLuc/705 cells containing an aberrant luciferase gene by using a quality-by-design approach. Critical formulation variables were linked to critical quality attributes (CQAs) using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). RESULTS: A series of CQAs were identified based on the quality target product profile. The L5 content and L5:Luc-ASO ratio (w/w) were determined as critical formulation variables, which were optimised systematically. The optimised Luc-ASO-loaded LPNs, defined from the OOS, displayed high loading and mediated splice correction at well-tolerated, lower doses as compared to those required for reference L5-based lipoplexes, L5-modified stable nucleic acid lipid nanoparticles or LPNs modified with dioleoyltrimethylammonium propane (conventional cationic lipid). CONCLUSIONS: The optimal Luc-ASO-loaded LPNs represent a robust formulation that mediates efficient intracellular delivery of Luc-ASO. This opens new avenues for further development of LPNs as a broadly applicable technology platform for delivering nucleic acid cargos intracellularly.

Identification of Factors of Importance for Spray Drying of Small Interfering RNA-Loaded Lipidoid-Polymer Hybrid Nanoparticles for Inhalation.

BACKGROUND: With the recent approval of the first small interfering RNA (siRNA) therapeutic formulated as nanoparticles, there is increased incentive for establishing the factors of importance for the design of stable solid dosage forms of such complex nanomedicines. METHODS: The aims of this study were: (i) to identify factors of importance for the design of spray-dried siRNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles (LPNs), and (ii) to evaluate their influence on the resulting powders by using a quality-by-design approach. Critical formulation and process parameters were linked to critical quality attributes (CQAs) using design of experiments, and an optimal operating space (OOS) was identified. RESULTS: A series of CQAs were identified based on the quality target product profile. The loading (ratio of LPNs to the total solid content) and the feedstock concentration were determined as critical parameters, which were optimized systematically. Mannitol was chosen as stabilizing excipient due to the low water content of the resulting powders. The loading negatively affected the colloidal stability of the LPNs, whereas feedstock concentration correlated positively with the powder particle size. The optimal mannitol-based solid formulation, defined from the OOS, displayed a loading of 5% (w/w), mass median aerodynamic diameter of 3.3 ± 0.2 µm, yield of 60.6 ± 6.6%, and a size ratio of 1.15 ± 0.03. Dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to non-spray-dried LPNs. CONCLUSION: The optimal solid dosage forms represent robust formulations suitable for higher scale-up manufacturing.