Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide.

In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.     

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 in vivo 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 (EC(50) ～ 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.     

Peptide Conjugates of Oligonucleotides As Enhanced Antisense Agents

The use of synthetic oligonucleotides and their analogs to block gene expression by binding the complementary RNA sequences in cells, the antisense principle, has been limited by poor uptake of the agents by cells in culture. This review describes attempts to harness by chemical conjugation the ability of certain peptides that may cross membranes to enhance the cellular uptake of oligonucleotides. These include fusogenic and hydrophobic peptides, nuclear localization signals, receptor targeting and translocating peptides, and various combinations. We also outline briefly some popular methods of peptide–oligonucleotide conjugation. Finally, we review the use of noncovalent peptide additives and the recent studies of conjugates of peptide nucleic acid (PNA) with peptides.     

Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption

Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.     

An efficient biodelivery system for antisense polyamide nucleic acid (PNA)

With the aim of developing a general and straightforward procedure for the intracellular delivery of naked peptide nucleic acids (PNAs), we designed an intracellularly biodegradable triphenylphosphonium (TPP) cation based transporter system. In this system, TPP is linked, via a biolabile disulfide bridge, to an activated mercaptoethoxycarbonyl moiety, allowing its direct coupling to the N-terminal extremity of a free PNA through a carbamate bond. We found that such TPP-PNA-carbamate conjugates were highly stable in a cell culture medium containing fetal calf serum. In a glutathione-containing medium mimicking the cytosol, the conjugates were rapidly degraded into an unstable intermediate, which spontaneously decomposed, releasing the free PNA. Using a fluorescence-labeled PNA-TPP conjugate, we demonstrated that conjugates were taken up by cells. Efficient cellular uptake and release of the PNA into the cytosol was further confirmed by the anti-HIV activity measured for the TPP-conjugate of a 16-mer PNA targeting the TAR region of the HIV-1 genome. This conjugate exhibited an IC(50) value of 1 microM, while the free 16-mer PNA did not inhibit replication of HIV in the same cellular test.     

Expression of nociceptin/OFQ receptor and prepro-nociceptin/OFQ in lymphoid tissues

This study was undertaken to examine the presence of functional nociceptin/orphanin FQ (N/OFQ) receptors in the immune system. Receptor mRNA signals were detected by RT-PCR in porcine thymus, lymph nodes, spleen and freshly-isolated splenocytes; the distribution of prepro-nociceptin/-orphanin FQ (PP-N/-OFQ) mRNA was similar, with the exception of lymph nodes. However, specific [³H]nociceptin binding sites were not detected in rat or porcine lymphoid tissues, and 0.1–100 nM nociceptin had no effect on forskolin-stimulated cyclic AMP concentrations in porcine splenocytes. Thus, it appears that nociceptin/orphanin FQ receptor mRNA, but not a functional receptor protein is expressed in the immune system.     

Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands

The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH₂, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis—Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5–8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.     

Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9

Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.     

Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli.

Peptide nucleic acid (PNA) is a DNA mimic with promising properties for the development of antisense agents. Antisense PNAs targeted to Escherichia coli genes can specifically inhibit gene expression, and attachment of PNA to the cell-permeabilizing peptide KFFKFFKFFK dramatically improves antisense potency. The improved potency observed earlier was suggested to be due to better cell uptake; however, the uptake kinetics of standard or modified PNAs into bacteria had not been investigated. Here we monitored outer and inner membrane permeabilization by using chemical probes that normally are excluded from cells but can gain access at points where membrane integrity is disturbed. Membrane permeabilization was much more rapid in the presence of peptide-PNA conjugates relative to the free components used alone or in combination. Indeed, peptide-PNAs permeabilized E. coli nearly as quickly as antimicrobial peptides. Furthermore, as expected for outer membrane-active compounds, added MgCl(2) reduced cell-permeabilization. Concurrent monitoring of outer and inner membrane permeabilization indicated that passage across the outer membrane is rate-limiting for uptake. The enhanced cell-permeation properties of peptide-PNAs can explain their potent antisense activity, and the results indicate an unanticipated synergy between the peptide and PNA components.     

Opioid receptor-like 1 (ORL1) receptor binding and the biological properties of Ac-Arg-Tyr-Tyr-Arg-Ile-Arg-NH2 and its analogs.

Hexapeptides such as Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) and Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH(2) have been isolated from a combinatorial peptide library as small peptide ligands for the opioid peptide-like 1 (ORL1) receptor. To investigate the detailed structural requirements of hexapeptides, 25 analogs of these hexapeptides, based on the novel analog Ac-Arg-Tyr-Tyr-Arg-Ile-Arg-NH(2) (1), were synthesized and tested for their ORL1 receptor affinity and agonist/antagonist activity on mouse vas deferens (MVD) tissues. Analog 1 and its Cit(6)-analog (10) were found to possess high affinity to the ORL1 receptor, comparable to that of nociceptin/orphanin FQ, and exhibited potent antagonist activity (pA(2) values of 7.77 for 1 and 7.51 for 10, which are higher than that of [NPhe(1)]nociceptin(1-13)-NH(2) (6.90) on MVD assay. It was also found that the amino acid residue in position 5 plays a key role in agonist/antagonist activity, i.e. an L-configuration aliphatic amino acid is required for potent antagonist activity, while a nonchiral or D-configuration residue produces potent agonist activity. These lines of evidence may provide insight into the mechanisms controlling agonist/antagonist switching in the ORL1 receptor, and may also serve to help developing more potent ORL1 agonists and antagonists.     

Inhibitory activity of nociceptin/orphanin FQ on capsaicin-induced bronchoconstriction in the guinea-pig

In vivo studies were conducted in the guinea-pig to investigate the activity of the selective ORL1 receptor agonist nociceptin/orphanin FQ against capsaicin-induced bronchoconstriction, a response mediated by the release of tachykinins from pulmonary sensory nerves. Anesthetized guinea-pigs were ventilated with a rodent ventilator and placed in a whole-body plethysmograph, and pulmonary resistance (R(L)) and dynamic lung compliance (C(Dyn)) were monitored. Intravenous administration of nociceptin/orphanin FQ (0.3 mg/kg) inhibited the capsaicin-induced bronchoconstriction. The new nonpeptide ORL1 receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) administered intravenously (1 mg/kg) produced a significant blockade of the inhibitory effect of nociceptin/orphanin FQ (0.3 mg/kg) on capsaicin-induced bronchoconstriction, whereas the nonselective opioid receptor antagonist naloxone (1 mg/kg) had no effect. Nociceptin/orphanin FQ (0.3 mg/kg) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist [beta-ala8]-neurokinin A (4-10). We conclude that nociceptin inhibits in vivo capsaicin-evoked tachykinin release from sensory nerve terminals in the guinea-pig by a prejunctional mechanism. This inhibitory action does not involve activation of opioid receptors.     

Nociceptin/Orphanin FQ Metabolism: Role of Aminopeptidase and Endopeptidase 24.15

Abstract: The endogenous opioid receptor-like₁ (ORL₁) ligand, nociceptin/orphanin FQ (FGGFTGARKSARKLANQ), a heptadecapeptide structurally resembling dynorphin A, has recently been identified. The wide distribution of ORL₁ mRNA and nociceptin/orphanin FQ precursor in the CNS, particularly in the limbic system regions and in several areas known to be involved in pain perception, suggests that nociceptin/orphanin FQ is potentially endowed with various central functions. In general, activation and/or inactivation of regulatory peptides occur through the action of cell surface peptidases. The physiological mechanisms under which nociceptin/orphanin FQ is metabolized should lead to a better understanding of its physiological functions. Mouse brain cortical slices were incubated in medium containing the heptadecapeptide in the presence or in the absence of peptidase inhibitors. The critical sites of enzymatic cleavage are Phe¹-Gly², Ala⁷-Arg⁸, Ala¹¹-Arg¹², and Arg¹²-Lys¹³ bonds. The major role played by metallopeptidases was confirmed by the complete protection of metabolism in the presence of EDTA. Aminopeptidase N and endopeptidase 24.15 are the two main enzymes involved in nociceptin/orphanin FQ metabolism, whereas endopeptidase 24.11 (involved in enkephalin [YGGFM(L)] catabolism) does not appear critically involved in nociceptin/orphanin FQ metabolism. The physiological relevance of aminopeptidase N and endopeptidase 24.15 in the heptadecapeptide metabolism remains to be determined.     

Design and synthesis of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-one as a novel class of GlyT1 inhibitors: achieving selectivity against the mu opioid and nociceptin/orphanin FQ peptide (NOP) receptors

A novel class of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-ones have been discovered and developed as potent and selective GlyT1 inhibitors. The molecules are devoid of activity at the GlyT2 isoform and display excellent selectivities against the mu opioid receptor as well as the nociceptin/orphanin FQ peptide (NOP) receptor. A novel, straightforward and efficient synthetic strategy for the assembly of the target molecules is also presented.     

Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway and therefore mechanisms that promote endosomal escape (or avoid the endosomal route) are required for improving bioavailability. A variety of auxiliary agents (chloroquine, calcium ions, or lipophilic photosensitizers) has this effect, but improved, unaided delivery would be highly advantageous in particular for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates that the lipophilic domain increases the endosomal uptake as well as promoting significantly endosomal escape. These results provide a novel route for improving the (cellular) bioavailability of larger hydrophilic molecules.     

Nociceptin/Orphanin FQ(1-13)NH2 analogues modified in the Phe1-Gly2 peptide bond

The synthesis and pharmacological activity of novel nociceptin/orphanin FQ (N/OFQ) analogues modified in the Phe(1)-Gly(2) peptide bond are reported. The aim of the present work was to elucidate the importance of this peptide bond for the N/OFQ receptor (NOP) interaction. Our study indicates that the first peptide bond in N/OFQ is important but not crucial for interaction with the N/OFQ receptor; for instance, substitution with a methyleneoxy bond generates an agonist derivative just 3-fold less potent than the reference compound.     

Designing bifunctional NOP receptor–mu opioid receptor ligands from NOP receptor-selective scaffolds. Part I.

The nociceptin receptor (NOP) and its endogenous agonist, nociceptin/orphanin FQ (N/OFQ), members of the opioid receptor and peptide families respectively, modulate the pharmacological effects of classical opioids, particularly opioid-induced reward and nociception. We hypothesized that compounds containing both NOP and opioid receptor activity in a single molecule may have useful pharmacological profiles as non-addicting analgesics or as drug abuse medications. We report here our forays into the structure–activity relationships for discovering ‘bifunctional’ NOP–mu opioid receptor (MOP) ligands, starting from our NOP-selective scaffolds. This initial SAR suggests pharmacophoric elements that may be modified to modulate/increase opioid affinity, while maintaining high affinity for the NOP receptor, to result in potent bifunctional small-molecule NOP/MOP ligands.     

Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.     

ANTIGENE PROPERTY OF PNA CONJUGATED TO THE NUCLEAR LOCALIZATION SIGNAL PEPTIDE

Peptide nucleic acid (PNA) is a DNA mimic with antigene properties. To enhance its capacity to enter in the cell and internalize in the nucleus, PNA has been conjugated to the nuclear localization signal (NLS) peptide, PKKKRKV. PNA-NLS conjugates form stable hybrids with complementary DNA strands and poorly tolerate mismatched base pairing. Employed against cancer-associated genes, PNA-NLS exhibited a potent and specific antigene activity, suggesting exciting therapeutic approaches to cancer.     

Designing bifunctional NOP receptor–mu opioid receptor ligands from NOP-receptor selective scaffolds. Part II

The nociceptin opioid receptor (NOP) and its endogenous peptide ligand nociceptin/orphanin FQ have been shown to modulate the pharmacological effects of the classical opioid receptor system. Suppression of opioid-induced reward associated with mu-opioid receptor (MOP)-mediated analgesia, without decreasing anti-nociceptive efficacy, can potentially be achieved with NOP agonists having bifunctional agonist activity at MOP, to afford ‘non-addicting’ analgesics. In Part II of this series, we describe a continuing structure–activity relationship (SAR) study of the NOP-selective piperidin-4-yl-1,3-dihydroindol-2-one scaffold, to obtain bifunctional activity at MOP, and a suitable ratio of NOP/MOP agonist activity that produces a non-addicting analgesic profile. The SAR reported here is focused on the influence of various piperidine nitrogen aromatic substituents on the ratio of binding affinity and intrinsic activity at both the NOP and MOP receptors.     

Synthesis and in vitro evaluation of PNA-peptide-DETA conjugates as potential cell penetrating artificial ribonucleases

We report the synthesis of novel artificial ribonucleases with potentially improved cellular uptake. The design of trifunctional conjugates 1a and 1b is based on the specific RNA-recognizing properties of PNA, the RNA-cleaving abilities of diethylenetriamine (DETA), and the peptide (KFF)(3)K for potential uptake into E. coli. The conjugates were assembled in a convergent synthetic route involving native chemical ligation of a PNA, containing an N-terminal cysteine, with the C-terminal thioester of the cell-penetrating (KFF)(3)K peptide to give 12a and 12b. These hybrids contained a free cysteine side-chain, which was further functionalized with an RNA-hydrolyzing diethylenetriamine (DETA) moiety. The trifunctional conjugates (1a, 1b) were evaluated for RNA-cleaving properties in vitro and showed efficient degradation of the target RNA at two major cleavage sites. It was also established that the cleavage efficiency strongly depended on the type of spacer connecting the PNA and the peptide.