Jerzy W. Jaroszewski

Antimicrobial, Hemolytic, and Cytotoxic Activities of β-Peptoid–Peptide Hybrid Oligomers: Improved Properties Compared to Natural AMPs

While natural host-defense antimicrobial peptides (AMPs) and analogues thereof have been investigated intensely in the last two decades with the purpose of combating the still increasing threat from emerging multiresistant pathogenic microbes, 2] compounds with peptidomimetic backbones have received considerable attention due to their superior stability against proteolytic enzymes. 4] Typically, studies of peptidomimetic AMP analogues have involved a brief microbiological evaluation of an array of oligomers, and only occasionally has testing been performed across a broader range of microorganisms or involved systematic structure–activity relationship (SAR) studies. Such investigations have proven fruitful for a-peptidic AMPs, 18] and might reveal unexpected lead structures and selectivity profiles when applied to peptidomimetics as well. Therefore, we have performed a more rigorous microbiological evaluation as well as toxicity profiling of a series of oligomers based on our b-peptoid–peptide hybrid backbone architecture. 20] Antimicrobial activities were determined alongside the archetypal cationic AMP magainin-2 and its clinically tested derivative pexiganan against a series of five important pathogens belonging to different classes. The obtained SAR data were subsequently correlated with various measurements of toxicity towards mammalian cells. Thereby we were able to derive useful trends for the future design of antibacterial and antifungal peptidomimetic constructs with potential for enhanced selectivity. Three subclasses 1 a–3 d (Scheme 1) were originally designed to address the general effects of length, type of cationic side chains, and presence of a-chirality in the b-peptoid residues. These series had previously been confirmed to possess membrane activity, as indicated by testing for hemolytic and prehemolytic effects, as well as by calcein release experiments with model liposomes, albeit the interaction of these compounds with intracellular targets cannot be ruled out based on our data. The all-aliphatic compound 4 and the mixed aromatic–aliphatic chimera 5 were included to address the importance of lipophilicity and type of cationic residue. Finally, we included three 5/6-carboxyfluorescein-labeled oligomers (6–8) to assess the influence of the presence of this widely used fluorophore on the antimicrobial activity, which might have important implications for confocal fluorescence microscopy studies of the interaction of labeled compounds with live bacteria. The chimeras 4–8 were of dodecamer length to minimize undesired mammalian cell toxicity that might be observed with increasing length. This compound collection was tested against a variety of clinically relevant pathogens and human red blood cells (Table 1). Scheme 1. Chemical structures of the examined hybrid oligomers. The abbreviations used for the b-peptoid units were adapted from the abbreviations commonly used for peptoids (i.e. , N-alkylglycines), 7] by adding the b-prefix. bNspe = N-(S)-1-phenylethyl-balanine, bNphe =b-N-phenylalanine, bNsce = N-(S)-1-cyclohexylethyl-b-alanine, hArg = homoarginine, CF = 5/6-carboxyfluoresceinoyl.

1H NMR spectroscopy-based interventional metabolic phenotyping: a cohort study of rheumatoid arthritis patients

1H NMR spectroscopy-based metabolic phenotyping was used to identify biomarkers in the plasma of patients with rheumatoid arthritis (RA). Forty-seven patients with RA (23 with active disease at baseline and 24 in remission) and 51 healthy subjects were evaluated during a one-year follow-up with assessments of disease activity (DAS-28) and 1H NMR spectroscopy of plasma samples. Discriminant analysis provided evidence that the metabolic profiles predicted disease severity. Cholesterol, lactate, acetylated glycoprotein, and lipid signatures were found to be candidate biomarkers for disease severity. The results also supported the link between RA and coronary artery disease. Repeated assessment using mixed linear models showed that the predictors obtained from metabolic profiles of plasma at baseline from patients with active RA were significantly different from those of patients in remission (P=0.0007). However, after 31 days of optimized therapy, the two patient groups were not significantly different (P=0.91). The metabolic profiles of both groups of RA patients were different from the healthy subjects. 1H NMR-based metabolic phenotyping of plasma samples in patients with RA is well suited for discovery of biomarkers and may be a potential approach for disease monitoring and personalized medication for RA therapy.

In Vitro Plasmodium falciparum Drug Sensitivity Assay: Inhibition of Parasite Growth by Incorporation of Stomatocytogenic Amphiphiles into the Erythrocyte Membrane

ABSTRACT Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 ± 0.5 μM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay.

Accelerated dereplication of crude extracts using HPLC–PDA–MS–SPE–NMR: Quinolinone alkaloids of Haplophyllum acutifolium

Direct hyphenation of analytical-scale high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) has been used for accelerated dereplication of crude extract of Haplophyllum acutifolium (syn. Haplophyllum perforatum). This technique allowed fast on-line identification of six quinolinone alkaloids, named haplacutine A-F, as well as of acutine, haplamine, eudesmine, and 2-nonylquinolin-4(1H)-one. Acutine and haplacutine E, isolated by preparative-scale HPLC, showed moderate antiplasmodial activity with IC(50) values of 2.17+/-0.22 microM and 3.79+/-0.24 microM, respectively (chloroquine-sensitive Plasmodium falciparum 3D7 strain).

Chemotaxonomy and evolution of Plantago L.

Abstract.In continuation of our investigations of the genus Plantago L. (Plantaginaceae), sixteen species were investigated with respect to water-soluble glycosides. The iridoids auroside, strictoloside and globularicisin, as well as poliumoside, 3-[(4-β-D-glucopyranosyloxy)phenyl]propionic acid and 2-[4-(β-D-glucopyranosyloxy)phenyl]acetic acid were isolated from Plantago for the first time. The latter compound has not previously been isolated as a natural product. Sorbitol was the main carbohydrate in all the species investigated. The distribution of iridoids correlates well with the morphological classification of Rahn and also with a recently published molecular phylogenetic study of nuclear ribosomal and plastid DNA sequences. A new chemotaxonomic finding is an abundance of iridoid glucosides present in one of the two groups within subgenus Coronopus section Coronopus, while the other group lacks iridoids, supporting a subdivision of this section. Moreover, in subgenus Albicans, 10-benzoylcatalpol is a characteristic constituent of section Gnaphaloides, while the corresponding cinnamoyl ester globularin occurs in section Lanceifolia. In biosynthetic experiments, labelled epideoxyloganic acid and deoxygeniposidic acid were incorporated into aucubin and geniposidic acid in Plantago ovata, consistent with earlier findings, but no incorporation into asperuloside was observed. The evolution of biosynthetic pathways in Plantago is discussed.

The Antiparasitic Compound Licochalcone A Is a Potent Echinocytogenic Agent That Modifies the Erythrocyte Membrane in the Concentration Range Where Antiplasmodial Activity Is Observed

ABSTRACT The well-known antiparasitic compound licochalcone A is a potent membrane-active agent that transforms normal erythrocytes into echinocytes in parallel with the inhibition of growth of Plasmodium falciparum cultures, the in vitro antiplasmodial effect apparently being an indirect effect on the host cell. In vitro experiments with synchronous cultures demonstrate that inhibition of invasion is the principal mechanism of growth inhibition. The erythrocyte membrane-modifying effect was also transiently observed in vivo in mice after intravenous administration.

Rational selection of antisense oligonucleotide sequences.

The purpose of this review is to identify rational selection procedures for the identification of optimal antisense oligonucleotide sequences. The review is firstly focused on how to find optimal hybridization sites, and secondly on how to select sequences that bind to structured RNA. The methods reviewed range from the more empirical testing of large numbers of mRNA complementary sequences to the more systematic techniques, i.e. RNase H mapping, use of combinatorial arrays and prediction of secondary structure of mRNA by computational methods. Structures that bind to structured RNA, i.e. aptastrucs and tethered oligonucleotide probes, and foldback triplex-forming oligonucleotides are also discussed. Relating to selection of antisense sequences by aid of computational analysis, valuable www addresses are given along with examples of folded structures of mRNA.

Combining HPLC-PDA-MS-SPE-NMR with circular dichroism for complete natural product characterization in crude extracts: levorotatory gossypol in Thespesia danis.

Despite recent demonstration of the power of HPLC-PDA-MS-SPE-NMR (high-performance liquid chromatography-photodiode-array detection-mass spectrometry-solid-phase extraction-nuclear magnetic resonance) in structure determination of natural products directly from minute amounts of crude extracts, this technique leaves chirality of the compounds uncharacterized. In this work we demonstrate that postcolumn SPE is a useful method of analyte concentration and accumulation not only for NMR but also for CD (circular dichroism) spectroscopy. Thus, use of HPLC-PDA-MS-SPE-NMR in combination with CD allowed rapid detection of ( R)-(-)-gossypol [( R)- 1] in Thespesia danis, providing a very rare example of the predominance of the levorotatory enantiomer of gossypol. Enantioselectivity of the in vitro antiplasmodial activity of gossypol was also demonstrated; the IC50 value of ( R)- 1 was 4.5 +/- 0.2 microM, with the eudismic ratio of about 2.5. No gossypol was detected in Gossypioides kirkii.

Cellular uptake and membrane-destabilising properties of α-peptide/β-peptoid chimeras: lessons for the design of new cell-penetrating peptides

Novel peptidomimetic backbone designs with stability towards proteases are of interest for several pharmaceutical applications including intracellular delivery. The present study concerns the cellular uptake and membrane-destabilising effects of various cationic chimeras comprised of alternating N-alkylated beta-alanine and alpha-amino acid residues. For comparison, homomeric peptides displaying octacationic functionalities as well as the Tat(47-57) sequence were included as reference compounds. Cellular uptake studies with fluorescently labelled compounds showed that guanidinylated chimeras were taken up four times more efficiently than Tat(47-57). After internalisation, the chimeras were localised primarily in vesicular compartments and diffusively in the cytoplasm. In murine NIH3T3 fibroblasts, the chimeras showed immediate plasma membrane permeabilising properties, which proved highly dependent on the chimera chain length, and were remarkably different from the effects induced by Tat(47-57). Finally, biophysical studies on model membranes showed that the chimeras in general increase the permeability of fluid phase and gel phase phosphatidylcholine (PC) vesicles without affecting membrane acyl chain packing, which suggests that they restrict lateral diffusion of the membrane lipids by interaction with phospholipid head groups. The alpha-peptide/beta-peptoid chimeras described herein exhibit promising cellular uptake properties, and thus represent proteolytically stable alternatives to currently known cell-penetrating peptides.

Recent advances in the medicinal chemistry of polyamine toxins.

This review describes the recent developments in the field of polyamine toxins, with focus on structure activity relationship investigations, including studies of importance of the polyamine moiety for biological activity, photolabeling studies using polyamine toxins as templates, as well as use of solid phase methods for the synthesis of polyamine toxins. The review is mainly concerned with effects of polyamine toxins on nicotinic acetylcholine receptors and ionotropic glutamate receptors.