Agmatine-grafted bioreducible poly(l-lysine) for gene delivery with low cytotoxicity and high efficiency.

Bioreducible cationic polymers have gained considerable attention in gene delivery due to their low cytotoxicity and high efficiency. In the present work, we reported a cationic polymer, poly(disulfide-l-lysine)-g-agmatine (denoted as SSL-AG), and evaluated its ability to transfer pEGFP-ZNF580 plasmid (pZNF580) into human umbilical vein endothelial cells (HUVECs). This SSL-AG polymeric carrier efficiently condensed pZNF580 into positively charged particles (<200 nm) through electrostatic interaction. This carrier also exhibited excellent buffering capacity in the physiological environment, good pDNA protection against enzymatic degradation and rapid pDNA release in a highly reducing environment mainly because of the responsive cleavage of disulfide bonds in the polymer backbone. The hemolysis assay and in vitro cytotoxicity assay suggested that the SSL-AG carrier and corresponding gene complexes possessed both good hemocompatibility and great cell viability in HUVECs. The cellular uptake of the SSL-AG/Cy5-oligonucleotide group was 3.6 times that of the poly(l-lysine)/Cy5-oligonucleotide group, and its mean fluorescence intensity value was even higher than that of the PEI 25 kDa/Cy5-oligonucleotide group. Further, the intracellular trafficking results demonstrated that the SSL-AG/Cy5-oligonucleotide complexes exhibited a high nucleus co-localization rate (CLR) value (36.0 ± 2.8%, 3.4 times that of the poly (l-lysine)/Cy5-oligonucleotide group, 1.6 times that of the poly(disulfide-l-lysine)-g-butylenediamine/Cy5-oligonucleotide group) at 24 h, while the endo/lysosomal CLR value was relatively low. This suggested that SSL-AG successfully delivered plasmid into HUVECs with high cellular uptake, rapid endosomal escape and efficient nuclear accumulation owing to the structural advantages of the bioreducible and agmatine groups. In vitro transfection assay also verified the enhanced transfection efficiency in the SSL-AG/pZNF580 group. Furthermore, the results of CCK-8, cell migration and in vitro/vivo angiogenesis assays revealed that pZNF580 delivered by SSL-AG could effectively enhance the proliferation, migration and vascularization of HUVECs. In a word, the SSL-AG polymer has great potential as a safe and efficient gene carrier for gene therapy.

Novel agmatine dipeptide inhibitors against the West Nile virus NS2B/NS3 protease: a P3 and N-cap optimization study.

This communication describes the synthesis and inhibitory activities of thirty-seven novel C-terminal agmatine dipeptides used as screening compounds to study the structure-activity relationship between active-site peptidomimetics and the West Nile virus (WNV) NS2B/NS3 serine protease. Our efforts lead to the discovery of a novel agmatine dipeptide inhibitor (compound 33, IC50 2.6 ± 0.3 µM) with improved inhibitory activity in comparison to the most potent inhibitor described in our recent report [IC50 4.7 ± 1.2 µM; Lim et al., Eur. J. Med. Chem. 46 (2011) 3130-3134]. In addition, our study cleared the contention surrounding the previous X-ray co-crystallization study and an enzyme inhibition report on the binding conformation adopted by active-site peptide aldehydes. Our data should provide valuable insights into the design of future peptidomimetic antivirals against WNV infections.

[Design, synthesis and Na+/H+ exchanger isoform-1 inhibitory activity of feruloylagmatine analogues].

In order to search for novel inhibitors of Na+/H+ exchanger isoform-1 (NHE-1), nine feruloylagmatine analogues were designed and synthesized from ferulic acid and agmatine. The structures of the synthesized compounds were confirmed by 1H NMR, 13C NMR and mass spectra, among which compounds 5f-5i were novel compounds. The results of preliminary pharmacological test showed that some of the compounds possessed strong NHE-1 inhibitory activity, among which compounds 5a, 5b and 6c were more potent than cariporide in NHE-1 inhibition.

Novel agmatine and agmatine-like peptidomimetic inhibitors of the West Nile virus NS2B/NS3 serine protease.

This communication reports the synthesis and inhibitory activities of novel non-covalent peptidomimetic inhibitors of the West Nile virus NS2B/NS3 protease containing a decarboxylated P1 arginine (agmatine; 4-aminobutylguanidine) and related analogues. One agmatine peptidomimetic (4-phenyl-phenacetyl-Lys-Lys-agmatine; compound 2) was shown to be a competitive inhibitor with a binding affinity of K(i) 2.05 ± 0.13 µM and was inactive against thrombin (IC(50) > 100 µM). Our results suggest that peptidomimetics with agmatine at the P1 position could potentially be employed as starting tools in the design of non-covalent competitive protease inhibitors due to their relative stability and ease of chemical synthesis compared to inhibitors containing reactive electrophilic warheads.

Amphoteric agmatine containing polyamidoamines as carriers for plasmid DNA in vitro and in vivo delivery.

In this paper we report on the investigation, as DNA nonviral carriers, of three samples of an amphoteric polyamidoamine bearing 4-aminobutylguanidine deriving units, AGMA5, AGMA10, and AGMA20, characterized by different molecular weights (M(w) 5100, 10100, and 20500, respectively). All samples condensed DNA in spherical, positively charged nanoparticles and protected it against enzymatic degradation. AGMA10 and AGMA20 polyplexes had average diameters lower than 100 nm. AGMA5 polyplexes were larger. All polyplexes showed negligible cytotoxicity and were internalized in cells. AGMA10 and AGMA20 performed differently from AGMA5 as nucleic acid carriers in vitro. AGMA10 and AGMA20 effectively promoted transfection, whereas AGMA5 was ineffective. FITC-labeled AGMA10 was prepared and the intracellular trafficking of its DNA polyplex was studied. DNA/AGMA10 polyplex was largely localized inside the nucleus, while AGMA10 concentrated in the perinuclear region. DNA/AGMA10 polyplex intravenously administered to mice promoted gene expression in liver but not in other organs without detectable toxic side effects.

Prevailingly cationic agmatine-based amphoteric polyamidoamine as a nontoxic, nonhemolytic, and "stealthlike" DNA complexing agent and transfection promoter.

AGMA1, a prevailingly cationic amphoteric polyamidoamine obtained by polyaddition of (4-aminobutyl)guanidine (agmatine) to 2,2-bis(acrylamido)acetic acid, was studied as a potential DNA carrier and transfection promoter. Fluorescein-labeled AGMA1 was prepared by conjugation with fluorescein isothiocyanate and its cell uptake, blood permanence, and body distribution studied. In spite of its cationic character, AGMA1 is neither toxic nor hemolytic in the pH range 4.0-7.4, circulates for a long time in the blood without preferentially localizing in the liver, easily enters HT-29 cells, gives stable complexes with DNA, and is endowed with good transfection efficiency, suggesting the ability to transport in the cytoplasm a DNA payload without any measurable membranolytic activity. If compared with other transfection promoters, including polyamidoamines of different structures, AGMA1 is apparently endowed with a unique combination of desirable requirements for a nonviral DNA polymer carrier and warrants potential as a transfection agent in vivo.

Synthesis and analgesic activity evaluation of some agmatine derivatives.

A series of N,N'-disubstituted-2-nitroethene-1,1-diamine and N,N'-disubstituted- N''-cyanoguanidine derivatives were prepared and evaluated for in vivo analgesic activity. The blood brain barrier (BBB) VolSurf model was used to predict the BBB permeation profiles of our synthesized compounds. Some compounds show both remarkable analgesic activity and good BBB permeation profiles, and these compounds might be developed for treatment of opioid tolerance and dependence.

[New charge-deficient agmatine analogs].

N,N'-Di-Boc-N"-triflylguanidine was demonstrated to be an efficient guanidinylation reagent for O-substituted hydroxylamines. N-(3-Aminooxypropyl)- and N-(3-aminopropoxy)guanidines, previously unknown isosteric and charge-deficient agmatine analogues, have been synthesized. The possibilities of using these compounds in studying polyamine metabolism are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

Novel hypotensive agents from Verbesina caracasana. 8. Synthesis and pharmacology of (3,4-dimethoxycinnamoyl)-N(1)-agmatine and synthetic analogues.

The more polar metabolites from the Venezuelan plant Verbesina caracasana, i.e., N(3)-prenylagmatine, (3,4-dimethoxycinnamoyl)-N(1)-agmatine, agmatine, and galegine (prenylguanidine), previously reported (Delle Monache, G.; et al. BioMed. Chem. Lett. 1999, 9, 3249-3254), have been synthesized following a biosynthetic strategy. The pharmacologic profiles of various synthetic analogues of (3,4-dimethoxycinnamoyl)-N(1)-agmatine (G5) were also analyzed, to shed some light on the structure-activity relationship of these compounds. Derivatives with the (E)-configuration and/or with a p-methoxybenzoyl moiety were found to be responsible for higher hypotensive effects, which were associated with a slight and, in some cases, not dose-related increase of cardiac inotropism, with variable and not significant chronotopic responses, and, only at higher doses, with effects of respiratory depression. Either an increase (to six) or a decrease (to two) of the number of methylene groups in the alkyl chain of (E)-G5 did not change blood pressure responses, while slightly increasing the positive inotropic ones. At pharmacological doses, all the studied compounds showed hypotensive and slight positive inotropic effects without relevant chronotropic and respiratory actions.

Agmatinase activity in rat brain: a metabolic pathway for the degradation of agmatine.

Agmatinase, the enzyme that hydrolyzes agmatine to form putrescine and urea in lower organisms, was found in rat brain. Agmatinase activity was maximal at pH 8-8.5 and had an apparent K(m) of 5.3 +/- 0.99 mM and a Vmax of 530 +/- 116 nmol/mg of protein/h. After subcellular fractionation, most of the enzyme activity was localized in the mitochondrial matrix (333 +/- 5 nmol/mg of protein/h), where it was enriched compared with the whole-brain homogenate (7.6-11.8 nmol/mg of protein/ h). Within the CNS, the highest activity was found in hypothalamus, a region rich in imidazoline receptors, and the lowest in striatum and cortex. It is interesting that other agmatine-related molecules such as arginine decarboxylase, which synthesizes agmatine, and I2 imidazoline receptors, for which agmatine is an endogenous ligand, are also located in mitochondria. The results show the existence of rat brain agmatinase, mainly located in mitochondria, indicating possible degradation of agmatine by hydrolysis at its sites of action.

N omega-hydroxyagmatine: a novel substance causing endothelium-dependent vasorelaxation.

Agmatine (Agm), a decarboxylated L-arginine, has been suggested to be an endogenous clonidine-displacing substance in the brain. We hypothesed that Agm enzymatically produces nitric oxide (NO) through N omega-hydroxyagmatine (OHAgm) similar to the well-known endogenous NO generation from L-arginine through N omega-hydroxy-L-arginine, because Agm possesses a guanidyl function in its molecule. OHAgm was originally synthesized from delta-aminopentanoic acid in 36% overall yield. Agm and the synthetic OHAgm were examined using rat aortic rings whether they could cause endothelium-dependent vasorelaxation or not. These substances equally elicited vasorelaxations. The relaxations were completely abolished by a NO synthase inhibitor, N omega-nitro-L-arginine methyl ester, or endothelium denudation. These results suggested that Agm and OHAgm are alternative precursors for NO generated by NOS and that OHAgm may be an endogenous substance distributable in the brain.

Competitive inhibition of a tumour cell surface protease. A rapid technique for in vitro testing of selective targeting systems.

The active centre of a protease on the surface of tumour cells can be located by its affinity for an active site-directed inhibitor, 9-amino acridine. Cells which have uninhibited proteases, bind 9-amino acridine and fluoresce in resin sections. The leukaemic rat was used as a model system to provide tumour cells in a well defined location. Drugs when coupled to a ligand (directed to the active centre of the protease) compete for this binding site with 9-amino acridine. Thus, competitive inhibition of the tumour cell surface protease provides a rapid technique for demonstrating the delivery of liganded molecules to the surface of tumour cells in vitro.

Synthesis of NG-monomethylagmatine, a decarboxylation product of NG-monomethyl-L-arginine.

NG-Monomethylagmatine, a decarboxylation product of NG-monomethyl-L-arginine, has been synthesized by reacting putrescine with N,S-dimethylthiopseudouronium iodide. The structural identity of the product was confirmed by proton NMR and mass spectroscopy, and its properties were determined on thin-layer and electrophoretic chromatography.

Insulin-like partial effects of agmatine derivatives in adipocytes.

In previous investigations, we described insulin-like effects of agmatine [(4-aminobutyl)guanidine] in vitro. In the present work we have examined whether these effects of agmatine can be enhanced by variation in chain length (C3 and C5 forms) and by alkylation. Propyl, butyl, pentyl, hexyl, octyl, isobutyl and isopentyl groups were introduced into C4- and C5-agmatine by hydrogenation of the corresponding azomethines. Alkylation of C3-agmatine was carried out by addition of alkylamines to acrylonitrile, followed by hydrogenation and amidination. For the biological assays, isolated fat cells from rat epididymic adipose tissue were used. N4-Butyl- and N4-pentyl-C4-agmatines lead to a two-fold, N4-hexyl-C4-agmatine to a three-fold enhancement of glucose oxidation in adipocytes. Alkylated C4- and C5-agmatines induce a three-fold increase in lipogenesis compared to agmatine. Alkylation of C3-agmatine does not increase its potency in this test. In our test system, insulin decreases adrenalin-induced lipolysis to 40% of the control value (100%). Agmatine and alkylated C4-agmatines yield very similar values (37% and 27-44% respectively). The alkylated C3-agmatines also exert strongly antilipolytic effects (25-35%), while the effects of the alkylated C5-agmatines are weaker. The synthesized agmatine derivatives were injected intraperitoneally into mice. Tolerable doses do not cause any significant reduction in blood glucose levels.