Synthesis and Evaluation of a Series of Bis(pentylpyridinium) Compounds as Antifungal Agents.

A series of bis(4-pentylpyridinium) compounds with a variety of spacers between the pyridinium headgroups was synthesised, and the antifungal activity of these compounds was investigated. Lengthening the alkyl spacer between the pentylpyridinium headgroups from 12 to 16 methylene units resulted in increased antifungal activity against C. neoformans and C. albicans, but also resulted in increased hemolytic activity and cytotoxicity against mammalian cells. However, inclusion of an ortho-substituted benzene ring in the centre of the alkyl spacer resulted in decreased cytotoxicity and hemolytic activity, while maintaining antifungal potency. Replacement of the alkyl and aromatic-containing spacers by more hydrophilic ethylene glycol groups resulted in a loss of antifungal activity. Some of the compounds inhibited fungal PLB1 activity, but the low correlation of this inhibition with antifungal potency indicates PLB1 inhibition is unlikely to be the predominant mode of antifungal action of this class of compounds, with preliminary studies suggesting they may act via disruption of fungal mitochondrial function.

In vitro antifungal activities of bis(alkylpyridinium)alkane compounds against pathogenic yeasts and molds.

Ten bis(alkylpyridinium)alkane compounds were tested for antifungal activity against 19 species (26 isolates) of yeasts and molds. We then determined the MICs and minimum fungicidal concentrations (MFCs) of four of the most active compounds (compounds 1, 4, 5, and 8) against 80 Candida and 20 cryptococcal isolates, in comparison with the MICs of amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin, using Clinical Laboratory and Standards Institutes broth microdulition M27-A3 (yeasts) or M38-A2 (filamentous fungi) susceptibility protocols. The compounds were more potent against Candida and Cryptococcus spp. (MIC range, 0.74 to 27.9 microg/ml) than molds (0.74 to 59.7 microg/ml). MICs against Exophiala were 0.37 to 5.9 microg/ml and as low as 1.48 microg/ml for Scedosporium but >or=25 microg/ml for zygomycetes, Aspergillus, and Fusarium spp. Compounds 1, 4, 5, and 8 exhibited good fungicidal activity against Candida and Cryptococcus, except for Candida parapsilosis (MICs of >44 mug/ml). Geometric mean (GM) MICs were similar to those of amphotericin B and lower than or comparable to fluconazole GM MICs but 10- to 100-fold greater than those for the other azoles. GM MICs against Candida glabrata were <1 microg/ml, significantly lower than fluconazole GM MICs (P<0.001) and similar to those of itraconazole, posaconazole, and voriconazole (GM MIC range of 0.4 to 1.23 microg/ml). The GM MIC of compound 4 against Candida guilliermondii was lower than that of fluconazole (1.69 microg/ml versus 7.48 microg/ml; P=0.012). MICs against Cryptococcus neoformans and Cryptococcus gattii were similar to those of fluconazole. The GM MIC of compound 4 was significantly higher for C. neoformans (3.83 mug/ml versus 1.81 microg/ml for C. gattii; P=0.015). This study has identified clinically relevant in vitro antifungal activities of novel bisalkypyridinium alkane compounds.

Synthesis, antifungal, haemolytic and cytotoxic activities of a series of bis(alkylpyridinium)alkanes.

A series of bis(alkylpyridinium)alkanes with a twelve carbon spacer between the positive charges was synthesised and their antifungal activity has been investigated. Compounds with 2-pentyl, 4-pentyl, 4-hexyl, 4-octyl, 4-propylbenzene, 3,4-dipentyl, 4-(5'-nonyl) and 3-methyl,4-pentyl head groups were the most potent antifungal agents with MICs in the range of 1.4-2.7 microM against reference strains of both Cryptococcus neoformans and Candida albicans.

Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing.

BACKGROUND: Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. RESULTS: The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate. CONCLUSION: The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.

In vitro interactions of tobramycin with various nonantibiotics against Pseudomonas aeruginosa and Burkholderia cenocepacia.

Pseudomonas aeruginosa and Burkholderia cepacia are the major pathogens that colonize the airway surface and cause progressive respiratory failure and high mortality, especially in cystic fibrosis (CF) patients. Tobramycin is the treatment of choice, but persistent usage enables the infectious organisms to activate defence mechanisms, making eradication rarely successful. Combinations of antibiotic and nonantibiotic compounds have been tested in vitro against P. aeruginosa and B. cepacia, but with mixed results. Sodium ions interfere with the bacterial tobramycin uptake system, but amiloride partially reverses this antagonism. In this pilot study, we extend previous findings of the effectiveness of tobramycin in combination with amiloride and other nonantibiotics against a P. aeruginosa type strain, and against four P. aeruginosa strains and one Burkholderia cenocepacia strain isolated from CF patients. Significantly, the four clinical P. aeruginosa strains were tobramycin resistant. We also find that Na+ and K+, but not Cl(-), are the chief antagonists of tobramycin efficacy. These results suggest that chemotherapy for CF patients might not only be compromised by antibiotic-resistant pathogens alone, but by a lack of penetration of antibiotics caused either by bacterial biofilms or the high sodium flux in the CF lung, or by antagonistic effects of some drug combinations, any of which could allow the persistence of drug-susceptible bacteria.

Synthesis, antifungal and antimicrobial activity of alkylphospholipids.

The antifungal, antibacterial and haemolytic activity of a series of alkylphosphocholines (e.g., miltefosine) and alkylglycerophosphocholines (e.g., edelfosine) has been investigated. These compound classes exhibit significant antifungal and moderate antibacterial activities. Several new alkylphosphocholine derivatives with amide or ester bonds in the alkyl chain have been synthesised. These compounds show much lower haemolytic activity than miltefosine. Alkylphosphocholines and alkylglycerophosphocholines show significant promise as novel orally available antifungal and antibacterial therapeutics.

Synthesis, antifungal and haemolytic activity of a series of bis(pyridinium)alkanes.

A series of bis(pyridinium)alkanes have been prepared and their antifungal activity, haemolytic activity and ability to inhibit fungal phospholipase B1 have been investigated, together with those of the commercially available antiseptics octenidine and dequalinium. Removal of the amino substituents from the pyridinium rings resulted in a significant decrease in antifungal activity. However, shortening or removing the alkyl chains attached to the amino groups had little effect on antifungal activity and significantly reduced haemolytic activity. Only octenidine was a strong inhibitor of fungal phospholipase B1.

In vitro activities of miltefosine and two novel antifungal biscationic salts against a panel of 77 dermatophytes.

The susceptibilities of 77 dermatophytes to miltefosine (MI), 1,12-bis(4-pentylpyridinium)dodecane (PYR), 1,12-bis(tributylammonium)dodecane (AM), itraconazole (ITC), terbinafine (TRB), and butenafine (BTF) were compared. Geometric mean MICs of TRB, BTF, ITC, MI, PYR, and AM were 0.039, 0.059, 1.718, 0.671, 6.006, and 4.771 microg/ml, respectively. MI was more active than ITC (P < 0.001).

Phospholipase B activity enhances adhesion of Cryptococcus neoformans to a human lung epithelial cell line.

Secreted phospholipase B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection by Cryptococcus neoformans and for dissemination from the lung via the lymphatics and blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (deltaplb1), and a reconstituted strain (deltaplb1rec). Adhesion of H99 and deltaplb1rec was approximately 69% greater than deltaplb1 at 4 h. Adhesion of deltaplb1 significantly increased after killing by chemicals or heat, and Fourier-transformed analysis by FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. Inhibition by specific PLB1 antibodies, or inhibitors of phospholipase B (PLB), but not lysophospholipase (LPL) or lysophospholipase transacylase (LPTA) activities decreased the adhesion of H99 and deltaplb1rec by 33-58%. Growth under conditions of osmotic stress and high glucose concentration increased both PLB secretion and subsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live deltaplb1 were observed in the presence of 0.1-2 mM palmitic acid. We conclude that PLB1 plays a role in the binding of C. neoformans to host lung epithelial cells, possibly due to production of fatty acids from plasma membranes and/or surfactant by PLB activity.

Correlation of antifungal activity with fungal phospholipase inhibition using a series of bisquaternary ammonium salts.

A series of bisquaternary ammonium salts with a 12-carbon spacer between the positive charges were synthesized, and their antifungal activity has been investigated. Compounds with butyl, pentyl, and isopentyl headgroups were the most potent antifungal agents with MICs in the range of 2.2-5.5 microM against both Cryptococcus neoformans and Candida albicans. The antifungal activity of these compounds correlated with their inhibition of cryptococcal phospholipase B1 (PLB1), a newly identified virulence factor. This indicates that the mode of action of these compounds may be inhibition of the fungal PLB1 enzyme, further validating this enzyme as a target for the development of novel antifungal therapies.

Hexadecylphosphocholine (miltefosine) has broad-spectrum fungicidal activity and is efficacious in a mouse model of cryptococcosis.

The alkyl phosphocholine drug miltefosine is structurally similar to natural substrates of the fungal virulence determinant phospholipase B1 (PLB1), which is a potential drug target. We determined the MICs of miltefosine against key fungal pathogens, correlated antifungal activity with inhibition of the PLB1 activities (PLB, lysophospholipase [LPL], and lysophospholipase-transacylase [LPTA]), and investigated its efficacy in a mouse model of disseminated cryptococcosis. Miltefosine inhibited secreted cryptococcal LPTA activity by 35% at the subhemolytic concentration of 25 microM (10.2 microg/ml) and was inactive against mammalian pancreatic phospholipase A2 (PLA2). At 250 microM, cytosolic PLB, LPL, and LPTA activities were inhibited by 25%, 51%, and 77%, respectively. The MICs at which 90% of isolates were inhibited (MIC90s) against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Cryptococcus gattii, Aspergillus fumigatus, Fusarium solani, Scedosporium prolificans, and Scedosporium apiospermum were 2 to 4 microg/ml. The MICs of miltefosine against Candida tropicalis (n = 8) were 2 to 4 microg/ml, those against Aspergillus terreus and Candida parapsilosis were 8 microg/ml (MIC90), and those against Aspergillus flavus (n = 8) were 2 to 16 microg/ml. Miltefosine was fungicidal for C. neoformans, with rates of killing of 2 log units within 4 h at 7.0 microM (2.8 microg/ml). Miltefosine given orally to mice on days 1 to 5 after intravenous infection with C. neoformans delayed the development of illness and mortality and significantly reduced the brain cryptococcal burden. We conclude that miltefosine has broad-spectrum antifungal activity and is active in vivo in a mouse model of disseminated cryptococcosis. The relatively small inhibitory effect on PLB1 enzyme activities at concentrations exceeding the MIC by 2 to 20 times suggests that PLB1 inhibition is not the only mechanism of the antifungal effect.

Cryptococcal phospholipases: a novel lysophospholipase discovered in the pathogenic fungus Cryptococcus gattii.

The pathogenic fungus Cryptococcus neoformans produces an extracellular PLB1 (phospholipase B1), shown previously to be a virulence factor. A novel phospholipase (LPL1) with only LPL (lysophospholipase) and LPTA (transacylase) activities has now been characterized in C. gattii, and found to be a 66-kDa glycoprotein (by SDS/PAGE), with a native molecular mass of 670 kDa. The pI was 6.3, and it was active at high temperatures (to 70 degrees C), as well as at both acidic and neutral pH values. It was stimulated by calcium and palmitoyl carnitine at pH 7.0, but not at pH 5.0, and palmitoyl lysophosphatidylcholine was the preferred substrate. Sequencing indicated that LPL1 is a novel cryptococcal lysophospholipase, and not the gene product of CnLYSO1 or PLB1. A protein with only LPL and LPTA activities was subsequently isolated from two strains of C. neoformans var. grubii. A PLB1 enzyme was isolated from both C. gattii and a highly virulent strain of C. neoformans var. grubii (H99). In both cases, all three enzyme activities (PLB, LPL and LPTA) were present in one 95-120 kDa glycoprotein (by SDS/PAGE) with pI 3.9-4.3. Characterization of PLB1 from C. gattii showed that it differed from that of C. neoformans in its larger native mass (275 kDa), high PLB activity relative to LPL and LPTA, and preference for saturated lipid substrates. Differences in the properties between the secreted phospholipases of the two cryptococcal species could contribute to phenotypic differences that determine their respective environmental niches and different clinical manifestations.

In vitro antifungal activities of inhibitors of phospholipases from the fungal pathogen Cryptococcus neoformans.

Secreted phospholipase B is a proven virulence factor for the pathogenic fungus Cryptococcus neoformans and exhibits three phospholipase activities in the one protein. These are phospholipase B (PLB), lysophospholipase (LPL), and lysophospholipase transacylase (LPTA). Our aim was to investigate the feasibility of using this enzyme as a target for antifungal therapy. We determined in C. neoformans var. grubii strain H99 that 82% of PLB activity was secreted but that 64% of LPL activity and 70% of LPTA activity were cell associated. Cell-associated activities (cytosolic and membrane) were further characterized, since it is likely that any fungicidal effect would depend on inhibition of these enzymes. Four commercially available compounds with structural similarities to phospholipid substrates were tested as inhibitors. These were alexidine dihydrochloride (compound A), dioctadecyldimethylammonium bromide (compound O), 1,12 bis-(tributylphosphonium)dodecane dibromide (compound P), and decamethonium dibromide (compound D). The best phospholipase inhibitors (compounds A and P) were also the most potent antifungal agents by the standard broth microdilution test. Compound A was highly selective for secreted and cell-associated PLB activities and showed no inhibition of mammalian phospholipase A(2) at 0.25 micro M. Compound O, which was specific for secretory and cytosolic LPL and LPTA and membrane-associated PLB, was not antifungal. We conclude that inhibitors of cryptococcal phospholipases can be selective for fungal enzymes and intrinsically antifungal. They also provide tools for assessing the relative importance of the various enzyme activities in virulence. Our results enable further rational structure-function studies to validate the use of phospholipases as antifungal targets.

The use of Tris-lipidation to modify drug cytotoxicity in multidrug resistant cells expressing P-glycoprotein or MRP1.

1. Increasing the lipophilicity is a strategy often used to improve a compound's cellular uptake and retention but this may also convert it into a substrate for an ATP-dependent transporter such as P-glycoprotein or the multidrug resistance-associated protein (MRP1), which are involved in cellular efflux of drugs. Tris-Lipidation of compounds is a convenient way of modifying drug lipophilicity and generating an array of derivatives with diverse properties. 2. To determine the effect of Tris-Lipidation on a drug's cytoxicity in multidrug resistant cells, various glycyl-Tris-mono- (GTP1), di- (GTP2) and tri-palmitate (GTP3) derivatives were prepared of the cancer chemotherapeutic drugs chlorambucil and methotrexate, and of the anti-HIV drug AZT. The cytotoxicity of these derivatives and their parent compounds was determined in the CEM/VLB(100) cells with increased P-glycoprotein expression, the CEM/E1000 cells that overexpress MRP1 and the parent, drug-sensitive CCRF-CEM cells. 3. Increasing the lipophilicity of AZT increased its cytotoxicity in the sensitive CCRF-CEM parental cell line while decreased cytotoxicity was observed for the methotrexate derivatives. For the chlorambucil derivatives, both increased (GTP1) and decreased (GTP2) cytotoxicity occurred in the CCRF-CEM cells. With the exception of AZT-GTP1, all GTP1 and GTP2 derivatives of chlorambucil, methotrexate and AZT had decreased cytotoxicity in the P-glycoprotein-expressing CEM/VLB(100) cells while chlorambucil-GTP1, methotrexate-GTP2 and methotrexate-GTP3 were the only compounds with decreased cytotoxicity in the MRP1-overexpressing CEM/E1000 cells. 4. The number of palmitate residues, the position of derivatisation and the type of linkage all may affect the P-glycoprotein and MRP1 substrate properties. 5. Tris-Lipidation may therefore provide a useful way of manipulating the pharmacokinetic properties of drugs.

Tris lipidation--a novel drug delivery system that alters biodistribution.

Tris-lipidation uses Tris to produce drug-fatty acyl conjugates. Radiolabelled Tris-fatty acyl conjugates of methotrexate (MTX) were examined in biodistribution studies in BALB/c mice. Following delivery via a variety of routes, the Tris-lipidated compounds demonstrated features in common with other colloid drug delivery systems. Tissues of the reticuloendothelial system localised the drug following intravenous administration, and the compounds showed prolongation at the site of injection into muscle or fatty tissue, subcutaneously or when inhaled. These findings indicate that the Tris-lipidation platform could be classed as an alternative colloid drug delivery system.