Nanomolar Cellular Antisense Activity of Peptide Nucleic Acid (PNA) Cholic Acid (“Umbrella”) and Cholesterol Conjugates Delivered by Cationic Lipids

Limited cellular uptake and low bioavailability of peptide nucleic acids (PNAs) have restricted widespread use of PNAs as antisense/antigene agents for cells in culture and not least for <i>in vivo</i> applications. We now report the synthesis and cellular antisense activity in cultured HeLa pLuc705 cells of cholesterol and cholic acid (“umbrella”) derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 μM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid, exhibited low nanomolar activity (EC₅₀ ∼ 25 nM). Excellent sequence specificity was retained, as mismatch PNA conjugates did not show any significant antisense activity. Furthermore, we show that increasing the transfection volume improved transfection efficiency, suggesting that accumulation (condensation) of the PNA/lipid complex on the cellular surface is part of the uptake mechanism. These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA–cholic acid conjugates which, in contrast to PNA–cholesterol conjugates, exhibit sufficient water solubility. The results also question the generality of using cholic acid “umbrella” derivatives as a delivery modality for antisense oligomers

DataSheet_1_Targeting synthesis of the Chromosome Replication Initiator Protein DnaA by antisense PNA-peptide conjugates in Escherichia coli.pdf

Initiation of chromosome replication is an essential stage of the bacterial cell cycle that is controlled by the DnaA protein. With the aim of developing novel antimicrobials, we have targeted the initiation of DNA replication, using antisense peptide nucleic acids (PNAs), directed against DnaA translation. A series of anti-DnaA PNA conjugated to lysine-rich bacterial penetrating peptides (PNA-BPPs) were designed to block DnaA translation. These anti-DnaA PNA-BPPs inhibited growth of wild-type Escherichia coli cells at low micromolar concentrations, and cells exposed to anti-DnaA PNA-BPPs exhibited characteristic hallmarks of chromosome replication inhibition. These results present one of very few compounds successfully targeting initiation of chromosome replication, an essential step in the bacterial cell cycle.</p

Role of SbmA in the Uptake of Peptide Nucleic Acid (PNA)-Peptide Conjugates in <i>E. coli</i>

Antisense PNA oligomers targeting essential genes (<i>acp</i>P or <i>fts</i>Z) and conjugated to the delivery peptide <i>L</i>((KFF)₃K) show complete growth inhibition of wild type <i>E. coli</i> strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA-peptide conjugates had disruptions in the region of <i>sbm</i>A, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with <i>sbm</i>A. Furthermore, deletion of <i>sbm</i>A in <i>E. coli</i> AS19, a strain that is sensitive to unmodified PNA, resulted in resistance to PNA. Finally, PNA conjugated with the corresponding non-biological H-<i>D</i>((KFF)₃K) peptide retained antibacterial activity in <i>sbm</i>A deletion strains, whereas the same conjugate with a protease-sensitive linker did not. These results clearly identify SbmA as a carrier of naked PNA over the inner bacterial membrane and thereby infer that the peptide is transporting the PNA conjugates over the outer membrane. Strains lacking SbmA were used to screen novel peptide-PNA carriers that were SbmA-independent. Four such PNA-peptide conjugates, H-<i>D</i>((KFF)₃K), H-(RFR)₄-Ahx-βAla, H-(R-Ahx-R)₄-Ahx-βAla, and H-(R-Ahx)₆-βAla, were identified that utilize an alternative uptake mechanism but retain their antimicrobial potency. In addition SbmA is the first protein identified to recognize PNA

Uranyl photocleavage of phosphorylated GFP28 analyzed by SDS PAGE and ESI-MS.

<p><b>A.</b> Lane 1–4: samples incubated for 30 minutes without irradiation in the presence of 0, 25, 50, 100 and 200 µM uranyl, respectively; Lane 5–7: samples irradiated for 30 min at 320 nm in the presence of 25, 50 and 100 µM uranyl, respectively; lane 8: cell lysate with GFP0 (no tag) <b>B.</b> Cleavage on ice. Lane 1: markers (35 and 45 kDa); lane 2: no irradiation, lane 3–5: irradiation on ice for 30 min in the presence of 25, 50 and 100 µM uranyl, respectively. <b>C.</b> Effect of pH. Lane 1: markers (35 and 45 kDa), lane 2: no irradiation; lane 3–8: cleavage in the presence of 25 µM uranyl at pH 9, 8.5, 8, 7.2, 6 and 4.5, respectively; lane 9: cell lysate with GFP0 (no tag) <b>D.</b> ESI-MS analysis of phosphorylated GFP28 cleavage. I: GFP treated with CK2; II and III: phosphorylated GFP28 irradiated (320 nm) at 50 and 100 µM uranyl, respectively.</p

Purification of GFP28 from <i>E. coli</i> protein extracts by uranyl-NTA agarose beads.

<p>SDS PAGE analysis by Coomassie brilliant blue staining. A.1: protein mixture (cleared cell lysate with GFP28, BSA and GFP0 treated with CK2); 2: supernatant after the beads are precipitated; 3–5: supernatant from washing steps; 6–7: eluates and 8: markers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091138#pone-0091138-g001" target="_blank">Figure 1</a>). <b>B.</b> protein mixture (cleared cell lysate with GFP28 treated with CK2); 2: supernatant after the beads are precipitated; 3–7: supernatant from washing steps; 8–9: eluates. Second round of purification: 10: supernatant after the beads are precipitated; 11–13: supernatant from washing steps; 14–15: eluates.</p

Antibacterial Peptide Nucleic Acid–Antimicrobial Peptide (PNA–AMP) Conjugates: Antisense Targeting of Fatty Acid Biosynthesis

Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knockdown of essential gene expression. However, discovery of efficient, nontoxic delivery vehicles for such PNA oligomers has remained a challenge. In the present study we show that antimicrobial peptides (AMPs) with an intracellular mode of action can be efficient vehicles for bacterial delivery of an antibacterial PNA targeting the essential <i>acpP</i> gene. The results demonstrate that buforin 2-A (BF2-A), drosocin, oncocin 10, Pep-1-K, KLW-9,13-a, (P59→W59)-Tat48–60, BF-2A-RXR, and drosocin-RXR are capable of transporting PNA effectively into <i>E. coli</i> (MICs of 1–4 μM). Importantly, presence of the inner-membrane peptide transporter SbmA was not required for antibacterial activity of PNA–AMP conjugates containing Pep-1-K, KLW-9,13-a, or drosocin-RXR (MICs of 2–4 μM)

Potent Inhibition of Late Stages of Hepadnavirus Replication by a Modified Cell Penetrating Peptide

<div><p>Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)₈ having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)₈ peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)₈ caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)₈-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis <em>via</em> interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)₈ may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.</p> </div

CPP sequences and IC50 of DHBV release.

<p>Sequences are shown in uppercase letters for peptides. The amino acids are <b>D</b> amino acids isomers. The carbohydrates chains are shown in lowercase letters. IC₅₀ represent the concentration of CPP resulting in 50% inhibition of DHBV release in PDH and LMH-D2 cells.</p

Inhibition of DHBV release in PDHs and LMH-D2 cells by modified cationic peptides and effect of Deca-(Arg)₈ on cell viability.

<p>Infected PDH and LMH-D2 cells were treated with 2 µM of several modified cationic peptides for 5 days. Cells supernatants were collected daily and were spotted onto positively charged nylon membrane and DHBV DNA was detected by hybridization with a DHBV DNA probe labeled with ³²P, and then was quantified by PhosphorImager scanning using ImageQuant software (Molecular Dynamics) to monitor DHBV release. Relative areas under curves determined by the means of duplicate, compared to untreated cells set at 100% are represented. The two representations show the effect of modified cationic peptides derived from (Arg)₈ sequence with increasing number of arginine in the peptide (<b>A</b>) or with increasing number of carbon in the fatty acid chain (<b>B</b>). Dose-dependent effects of Deca-(Arg)₈ transduction on cell viability (<b>C</b>). Cell viability was determined by enzymatic activity MTT assay after daily incubation with different concentrations of Deca-(Arg)₈ ranking from 1 µM to 4 µM during six days. The error bars display the standard deviation of duplicates in three independent experiments.</p

Dose dependent inhibition of hepadnaviral release by Deca-(Arg)₈ in different cell culture systems.

<p>DHBV-infected PDHs (<b>A</b>), and stable transfected LMH-D2 cells (<b>B</b>) were treated with increasing amounts of Deca-(Arg)₈ ranking from 0.25 µM to 2 µM in duplicates for 6 and 4 days, respectively. Cell culture supernatants were collected daily during treatment. The kinetics of viral release in cell culture supernatants, monitored by dot-blot hybridization and quantified by PhosphorImager scanning using ImageQuant software (Molecular Dynamics) is represented on the upper panel. Relative areas under curves determined by the means of duplicate, compared to untreated cells, are represented in the lower panel. The curves are representative of at least two independent experiments.</p