Peptide dendrimers as antifungal agents and carriers for potential antifungal agent-N3 -(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid-synthesis and antimicrobial activity.

A series of peptide dendrimers and their conjugates with antimicrobial agent FMDP (N3 -(4-methoxyfumaroyl)-(S)-2,3-diamino-propanoic acid) were synthesized. The obtained compounds were tested for the antibacterial and antifungal activity. All novel dendrimers displayed much better activity against the tested strains than FMDP itself. Moreover, their conjugates with FMDP also exhibited antimicrobial activity. The most promising molecules were tested against a broad selection of fungal strains. The analysis of their antifungal properties indicates that the examined molecules are efficient growth inhibitors of fluconazole-resistant hospital-acquired strains. Moreover, an application of amphiphilic branched peptides such as FMDP carriers suggests that transport mechanism involves more likely the cell membrane perturbation than the mediation of the specific transport proteins. The activity of obtained compounds strongly depends on the specific structure of the molecule.

Emerging Anticancer Activity of Candidal Glucoseamine-6-Phosphate Synthase Inhibitors upon Nanoparticle-Mediated Delivery.

Numerous glutamine analogues have been reported as irreversible inhibitors of the glucosamine-6-phosphate (GlcN-6-P) synthase in pathogenic Candida albicans in the last 3.5 decades. Among the reported inhibitors, the most effective N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP) has been extensively studied in order to develop its more active analogues. Several peptide-FMDP conjugates were tested to deliver FMDP to its subcellularly located GlcN-6-P synthase target. However, the rapid development of fungal resistance to FMDP-peptides required development of different therapeutic approaches to tackle antifungal resistance. In the current state of the global antifungal resistance, subcellular delivery of FMDP via free diffusion or endocytosis has become crucial. In this study, we report on in vitro nanomedical applications of FMDP and one of its ketoacid analogues, N3- trans-4-oxo-4-phenyl-2-butenoyl-l-2,3-diaminopropanoic acid (BADP). FMDP and BADP covalently attached to polyethylene glycol-coated iron oxide/silica core-shell nanoparticles are tested against intrinsically multidrug-resistant C. albicans. Three different human cancer cell lines potentially overexpressing the GlcN-6-P synthase enzyme are tested to demonstrate the immediate inhibitory effects of nanoparticle conjugates against mammalian cells. It is shown that nanoparticle-mediated delivery transforms FMDP and BADP into strong anticancer agents by inhibiting the growth of the tested cancer cells, whereas their anti-Candidal activity is decreased. This study discusses the emerging inhibitory effect of the FMDP/BADP-nanoparticle conjugates based on their cellular internalization efficiency and biocompatibility.

Acetate-Induced Disassembly of Spherical Iron Oxide Nanoparticle Clusters into Monodispersed Core-Shell Structures upon Nanoemulsion Fusion.

It has been long known that the physical encapsulation of oleic acid-capped iron oxide nanoparticles (OA-IONPs) with the cetyltrimethylammonium (CTA+) surfactant induces the formation of spherical iron oxide nanoparticle clusters (IONPCs). However, the behavior and functional properties of IONPCs in chemical reactions have been largely neglected and are still not well-understood. Herein, we report an unconventional ligand-exchange function of IONPCs activated when dispersed in an ethyl acetate/acetate buffer system. The ligand exchange can successfully transform hydrophobic OA-IONP building blocks of IONPCs into highly hydrophilic, acetate-capped iron oxide nanoparticles (Ac-IONPs). More importantly, we demonstrate that the addition of silica precursors (tetraethyl orthosilicate and 3-aminopropyltriethoxysilane) to the acetate/oleate ligand-exchange reaction of the IONPs induces the disassembly of the IONPCs into monodispersed iron oxide-acetate-silica core-shell-shell (IONPs@acetate@SiO2) nanoparticles. Our observations evidence that the formation of IONPs@acetate@SiO2 nanoparticles is initiated by a unique micellar fusion mechanism between the Pickering-type emulsions of IONPCs and nanoemulsions of silica precursors formed under ethyl acetate buffered conditions. A dynamic rearrangement of the CTA+-oleate bilayer on the IONPC surfaces is proposed to be responsible for the templating process of the silica shells around the individual IONPs. In comparison to previously reported methods in the literature, our work provides a much more detailed experimental evidence of the silica-coating mechanism in a nanoemulsion system. Overall, ethyl acetate is proven to be a very efficient agent for an effortless preparation of monodispersed IONPs@acetate@SiO2 and hydrophilic Ac-IONPs from IONPCs.

Synthesis and biological activity of novel ester derivatives of N(3)-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid containing amide and keto function as inhibitors of glucosamine-6-phosphate synthase.

A short series of novel ester derivatives of N(3)-4-metoxyfumaroyl-(S)-2,3-diaminopropanoic acid (FMDP) containing amide or keto functions have been designed and synthesized. Their antifungal activity and inhibitory properties toward fungal glucosamine-6-phosphate synthase has also been evaluated. The obtained compounds 11-13 and 15-17 demonstrated good antifungal activity against Candida albicans. Compounds 11-13 displayed also potent inhibitory activity against fungal glucosamine-6-phosphate synthase, comparable to that of FMDP.

Synthesis, anticandidal activity of N(3)-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic amide derivatives--novel inhibitors of glucosamine-6-phosphate synthase.

Novel FMDP amides 4-6 have been synthesized and tested against Candida strains. The anticandidal activity has been confined only to Candida albicans. Anticandidal activity of the tested amides has correlated with their inhibitory activity of glucosamine-6-phosphate synthase in cell free extract from C. albicans.

Inactivation of glucosamine-6-phosphate synthase by N3-oxoacyl derivatives of L-2,3-diaminopropanoic acid.

N(3)-Oxoacyl derivatives of L-2,3-diaminopropanoic acid 1-4, containing either an epoxide group or a conjugated double bond system, inactivate Saccharomyces cerevisiae glucosamine-6-phosphate (GlcN-6-P) synthase in a time- and concentration dependent manner. The results of kinetics studies on inactivation suggested a biphasic course, with formation of the enzyme-ligand complex preceding irreversible modification of the enzyme. The examined compounds differed markedly in their affinity to the enzyme active site. Inhibitors containing a phenyl ketone moiety bound much more strongly than their methyl ketone counterparts. The molecular mechanism of enzyme inactivation by phenyl ketone compounds 1 and 3 was elucidated by using a stepwise approach with 2D NMR, MS and UV-visible spectroscopy. A substituted thiazine derivative was identified as the final product of a model reaction between an epoxide compound, 1, and L-cysteine ethyl ester (CEE); and the respective cyclic product, found as a result of reaction between 1 and CGIF tetrapeptide, was identical to the N-terminal fragment of GlcN-6-P synthase. On the other hand, the reaction of a double-bond-containing compound, 3, with CEE, CGIF and GlcN-6-P synthase led to the formation of a C-S bond, without any further conversion or rearrangement. Molecular mechanisms of the reactions studied are proposed.

The synthesis and biological activity of lipophilic derivatives of bicine conjugated with N³-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP)-an inhibitor of glucosamine-6-phosphate synthase.

A series of bis-N,N-(2-hydroxyethyl)glycine (bicine) derivatives, conjugated with an inhibitor of glucosamine-6-phosphate synthase, have been synthesized and their lipophilic and antifungal properties have been tested. The obtained compounds demonstrated higher lipophilicity than free inhibitor (FMDP) and, in consequence, an increased potential to cross the cytoplasmic membrane. All the tested compounds show better antifungal activity than parent compound.

New imidazoline/α(2)-adrenoceptors affecting compounds-4(5)-(2-aminoethyl)imidazoline (dihydrohistamine) derivatives. Synthesis and receptor affinity studies.

Compilation of agmatine structure and imidazoline moiety leads to a new group of imidazoline/α(2)-adrenoceptor ligands, 4(5)-(2-aminoethyl)imidazoline derivatives. In this study the exploration of previously unknown 4(5)-(2-aminoethyl)imidazolines including the analogues of reported imidazoline and α(2)-aderenoceptors ligands: clonidine, rilmenidine, idazoxan, efaroxan, antazoline, tracizoline is described. The synthesis of a variety of novel 4(5)-(2-aminoethyl)imidazolines and their I(1), I(2), α(2)-adrenoceptors affinities are reported.

Role for chitin and chitooligomers in the capsular architecture of Cryptococcus neoformans.

Molecules composed of beta-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformans varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformans cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformans in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.

New analogues of agmatine with higher affinity to imidazoline receptors.

Compilation of agmatine structure and imidazoline ring leads to a new family of imidazoline/alpha(2)-adrenoceptor ligands, 4(5)-(2-aminoethyl)imidazoline derivatives. Constraining of the guanidine moiety into heterocyclic ring improved the affinities of the resultant fusion compounds in comparison to agmatine itself. In this work, the synthetic approach and results for I(1), I(2), and alpha(2)-adrenoceptors affinities are reported.

Antifungal activity of thionated analogues of Nva-FMDP and Lys-Nva-FMDP.

The antifungal activity of synthetic endothiopeptdides, i.e. Nva(psi)[CSNH]-FMDP and Lys(psi)[CSNH]-Nva(psi)[CSNH]-FMDP was studied in medium containing blood serum, against selected Candida strains; Candida albicans Gu4 (fluconazole sensitive), C. albicans Gu5 (fluconazole resistant), C. albicans ATCC 10231, Candida krusei DSM 6128 and Candida parapsilosis DSM 5784. Although thiopeptide bonds in the tested peptides increased their stability in blood serum, their antifungal activity, however, drastically decreased in comparison with the peptides containing non-modified peptide bonds. Moreover, the inhibitory activity towards glucosamine-6-phosphate synthase of thionated synthetic analogue of FMDP was performed. The thiopeptdide bond also influenced its inhibitory properties against enzyme from C. albicans.

Enzymatic degradation and transport of endothiopeptides into Escherichia coli K12 mutant strains.

Transport of three synthesized endothiopeptides: AlaPsi[CSNH]Ala, AlaPsi[CSNH]Leu, and AlaPsi[CSNH]Phe, into Escherichia coli K12 mutant strains and enzymatic degradation studies were carried out. These compounds, well transported by permeases, but significantly more resistant to enzymatic cleavage than the corresponding natural peptides, seem to be useful as enzyme inhibitor carriers.

The immunoregulatory effects of edeine analogues in mice.

The edeines analogs were tested in several in vitro and in vivo assays using the mouse model, with edeine B (peptide W1) and cyclosporine A as reference compounds. The peptides displayed moderate, stimulatory effects on concanavalin A-induced (ConA-induced) splenocyte proliferation, whereas their effects on pokeweed mitogen-induced (PWM-induced) splenocyte proliferation were inhibitory. The peptides inhibited lipopolysacharide-induced (LPS-induced) tumor necrosis factor alpha production but had little effect on interleukin 6 production. In the model of the humoral immune response in vitro to sheep red blood cells, peptide 1 was distinctly stimulatory in the investigated concentrations (1-100 microg/ml), whereas peptides 3 and 4 only stimulated the number of antibody-forming cells at the highest concentration (100 microg/ml). In the model of the delayed type hypersensitivity in vivo to ovalbumin, the peptides were moderately suppressive (3 being the most active). The reference peptide W1 stimulated ConA-induced cell proliferation at 1-10 microg/ml but was inhibitory at 100 microg/ml. It also inhibited PWM-induced cell proliferation in a dose-dependent manner. This peptide had no effect on the humoral immune response in vitro or on cytokine production, but inhibited DTH reaction in vivo. The relationship between structure and activity, and a possible mode of action of the peptides, is discussed in this paper.

Structure activity relationship studies on the antimicrobial activity of novel edeine A and D analogues.

Edeines are pentapeptide amide antibiotics composed of four nonprotein amino acids, glycine, and polyamine. They exhibit antimicrobial and immunosuppressive activities and are universal inhibitors of translation. Moreover, it was proven that the free ionizable carboxy group in the (2R, 6S, 7R)-2,6-diamino-7-hydroxyazelaic acid moiety is not essential for biological activity of these compounds. In this paper we describe the synthesis of four novel edeine A and D analogues in which the above-mentioned acid residue was replaced with the (3R, 4S)- or (3S, 4S)-4,5-diamino-3-hydroxypentanoic acid moiety. In one compound we also introduced into molecule the 3-N,N-dimethyl derivative of (S)-2,3-diaminopropanoic acid to prevent the transpeptidation process, which results in the loss of biological activity of alpha-isomers of edeines. All peptides were synthesized applying the active ester and azide methods and on the basis of the coupling of suitable N-terminal tripeptides with proper C-terminal dipeptide amides. The activities of the newly obtained edeine analogues against selected strains of bacteria and fungi are also presented.

Glutamine analogues containing a keto function--novel inhibitors of fungal glucosamine-6-phosphate synthase.

A series of novel inhibitors of glucosamine-6-phosphate synthase, analogues of AADP and BADP, have been synthesized and their inhibitory, lipophilic and antifungal properties have been tested. The improvement in lipophilicity has not much affected the antifungal activity of the new compounds. Dipeptides containing norvaline and selected inhibitors have shown substantial activity against S. cerevisiae and C. glabrata and only poor activity against C. albicans strain. These peptides do not seem to be toxic towards human cells.

Anticandidal properties of N-acylpeptides containing an inhibitor of glucosamine-6-phosphate synthase.

A series of N-acyl peptides 1-9, containing an inhibitor of glucosamine-6-phosphate synthase have been synthesised and tested against Candida strains. N-Acylated peptides inhibit glucosamine-6-phosphate synthase in cell free extracts from Candida albicans. Antifungal activities of the tested compounds correlated with their lipophilic properties. Peptides acylated with decanoic acid were found to be the most potent in the series. N-decanoylpeptides also showed activity against Candida albicans Gu5 resistant mutant with Cdr1 and Cdr2 drug extrusion proteins that causes MDR by an active efflux mechanism.

Structure-activity relationships for a series of peptidomimetic antimicrobial prodrugs containing glutamine analogues.

Synthetic glutamine analogues such as N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP) inhibit purified glucosamine-6-phosphate synthase, an intracellular enzyme that is essential for microbial cell wall synthesis, but they are inactive against intact organisms because they cannot enter the cell. However, when the analogues are linked to a peptide they can be actively transported, and FMDP peptidomimetics show broad-spectrum antimicrobial activity. To characterize this process in more detail, the antibacterial activities of various synthetic peptidomimetics containing glutamine analogues have been determined against isogenic strains of Escherichia coli in which one or more of its three peptide transporters Dpp, Opp and Tpp have been mutated. In addition, their affinities for DppA and OppA, the binding-protein components of the transporters, have been measured. In general, antibacterial activities against the various transport mutants correlated with binding to DppA and OppA. Xaa-FMDP compounds have greater activities than FMDP-Xaa analogues. To explore structure-activity relationships for the peptidomimetics, molecular modelling was used to determine the conformational forms they adopt in solution. The relative bioactivities of the peptidomimetics correlated with the percentage of conformers that had backbone torsions matching those previously defined for the molecular recognition templates of the peptide transporters. However, the large size of the N-terminal residue in the FMDP-Xaa analogues appears to interfere with transport and thus to limit antibacterial activity. Overall, the results provide the structural rationale for the identification in silico of analogues with optimal bioactivities, which decreases the need for extensive chemical syntheses and testing.

Antibacterial action of dipeptides containing an inhibitor of glucosamine-6-phosphate isomerase.

Several dipeptides, containing the N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) moiety linked to protein and non-protein amino acids, exhibited a strong growth-inhibitory and bactericidal effect against Bacillus subtilis. FMDP-dipeptides were efficiently transported into bacterial cells by a di-tripeptide permease and subsequently cleaved by intracellular Mn2+/Co2+-dependent peptidases. Cleavage rates [0.1-5.6 micromol min-1 (mg protein)-1] were about two orders of magnitude lower than transport rates [40-200 micromol min-1 (mg dry wt)-1]. The released FMDP inactivated glucosamine-6-phosphate (GlcN-6-P) isomerase, an enzyme catalysing the first committed step in a biosynthetic pathway leading to amino sugar-nucleotide precursors of bacterial peptidoglycan. Inhibition of GlcN-6-P isomerase precluded peptidoglycan biosynthesis and resulted in a strong bacteriolytic effect. Results of the studies on consequences of GlcN-6-P isomerase inhibition upon the action of FMDP-dipeptides provided evidence demonstrating that the lack of endogenous GlcN-6-P could be a reason for the triggering of bacterial autolysis. Peptides containing the inhibitors of GlcN-6-P isomerase are one of the very few antimicrobial agents known that exhibit both bactericidal and fungicidal effects.