Synthesis of kanamycin-azole hybrids and investigation of their antifungal activities.

The World Health Organization (WHO) recognizes Candida albicans and Cryptococcus neoformans as the critical priority fungal pathogens for which therapeutic solutions are needed. Azole-based antifungal agents, including triazoles, diazoles, and thiazoles, are widely used in the treatments for fungal infections. In light of past successes in the transformation of antibacterial kanamycin into antifungal derivatives via chemical modifications, a new library of kanamycin-azole hybrids was synthesized and tested against a panel of azole-resistant and susceptible Candida and Cryptococcus strains.

Scalable and cost-effective tosylation-mediated synthesis of antifungal and fungal diagnostic 6″-Modified amphiphilic kanamycins.

Amphiphilic kanamycins bearing hydrophobic modifications at the 6″ position have attracted interest due to remarkable antibacterial-to-antifungal switches in bioactivity. In this report, we investigate a hurdle that hinders practical applications of these amphiphilic kanamycins: a cost-effective synthesis that allows the incorporation of various connecting functionalities to which the hydrophobic moieties are connected to the kanamycin core. A cost-effective tosylation enables various modifications at the 6″ position, which is scalable to a 90-g scale.

Antifungal Activities of 4″,6″-Disubstituted Amphiphilic Kanamycins.

Amphiphilic kanamycins derived from the classic antibiotic kanamycin have attracted interest due to their novel bioactivities beyond inhibition of bacteria. In this study, the recently described 4″,6″-diaryl amphiphilic kanamycins reported as inhibitors of connexin were examined for their antifungal activities. Nearly all 4″,6″-diaryl amphiphilic kanamycins tested had antifungal activities comparable to those of 4″,6″-dialkyl amphiphilic kanamycins, reported previously against several fungal strains.

Development of Fungal Selective Amphiphilic Kanamycin: Cost-Effective Synthesis and Use of Fluorescent Analogs for Mode of Action Investigation.

Amphiphilic aminoglycosides have attracted interest due to their novel antifungal activities. A crucial but often neglected factor for drug development in academia is cost of production. Herein is reported a one-step, inexpensive synthesis of amphiphilic alkyl kanamycins constituted with only natural components.

Synthesis and biological activity investigation of azole and quinone hybridized phosphonates.

Phosphonates, azoles and quinones are pharmacophores found in bioactive compounds. A series of phosphonates conjugated to azoles and quinones with variable carbon chain lengths were synthesized in 3-4 steps with good yield. Antifungal assay of these compounds showed that ethyl protected phosphates have excellent inhibitory activity against phytopathogenic fungus Fusarium graminearum, and the free-base phosphates have good activity against human pathogenic fungi Aspergillus flavus and Candida albicans.

One-step synthesis of carbohydrate esters as antibacterial and antifungal agents.

Carbohydrate esters are biodegradable, and the degraded adducts are naturally occurring carbohydrates and fatty acids which are environmentally friendly and non-toxic to human. A simple one-step regioselective acylation of mono-carbohydrates has been developed that leads to the synthesis of a wide range of carbohydrate esters. Screening of these acylated carbohydrates revealed that several compounds were active against a panel of bacteria and fungi, including Staphylococcus aureus, methicillin-resistant S.

In vitro antifungal synergy between amphiphilic aminoglycoside K20 and azoles against Candida species and Cryptococcus neoformans.

Several azoles are widely used to treat human fungal infections. Increasing resistance to these azoles has prompted exploration of their synergistic antifungal activities when combined with other agents. The amphiphilic aminoglycoside, K20, was recently shown to inhibit filamentous fungi, yeasts and heterokonts, but not bacteria.

Antifungal amphiphilic aminoglycoside K20: bioactivities and mechanism of action.

K20 is a novel amphiphilic antifungal aminoglycoside that is synthetically derived from the antibiotic kanamycin A. Reported here are investigations of K20's antimicrobial activities, cytotoxicity, and fungicidal mechanism of action. In vitro growth inhibitory activities against a variety of human and plant pathogenic yeasts, filamentous fungi, and bacteria were determined using microbroth dilution assays and time-kill curve analyses, and hemolytic and animal cell cytotoxic activities were determined.

Production of destruxins from Metarhizium spp. fungi in artificial medium and in endophytically colonized cowpea plants.

Destruxins (DTXs) are cyclic depsipeptides produced by many Metarhizium isolates that have long been assumed to contribute to virulence of these entomopathogenic fungi. We evaluated the virulence of 20 Metarhizium isolates against insect larvae and measured the concentration of DTXs A, B, and E produced by these same isolates in submerged (shaken) cultures. Eight of the isolates (ARSEF 324, 724, 760, 1448, 1882, 1883, 3479, and 3918) did not produce DTXs A, B, or E during the five days of submerged culture.

Membrane lipid-modulated mechanism of action and non-cytotoxicity of novel fungicide aminoglycoside FG08.

A novel aminoglycoside, FG08, that differs from kanamycin B only by a C8 alkyl chain at the 4″-O position, was previously reported. Unlike kanamycin B, FG08 shows broad-spectrum fungicidal but not anti-bacterial activities. To understand its specificity for fungi, the mechanism of action of FG08 was studied using intact cells of the yeast Saccharomyces cerevisiae and small unilamellar membrane vesicles.

Sphingolipid C4 hydroxylation influences properties of yeast detergent-insoluble glycolipid-enriched membranes.

Sphingoid base C4 hydroxylation is required for syringomycin E action on the yeast plasma membrane. Detergent-insoluble glycolipid-enriched membranes (DIGs) from a yeast strain lacking C4 hydroxylated sphingoid bases (sur2delta) are composed of linear membrane fragments instead of vesicular structures observed for wild-type DIGs, though they have similar lipid compositions and amounts of DIG marker proteins. Light-scattering bands collected from sur2delta after centrifugation of Triton X-100-treated cell lysates in continuous density gradients have lower buoyant densities than that of the wild-type.

Structure-function studies of yeast C-4 sphingolipid long chain base hydroxylase.

The roles of putative active site residues of the Saccharomyces cerevisiae sphingolipid C-4 long chain base hydroxylase (Sur2p) were investigated by site-directed mutagenesis. The replacement of any one of conserved His residues of three histidine-rich motifs with an alanine eliminated hydroxylase activity in vivo and in vitro, indicating that they are all essential elements of the active site. An additional conserved His residue (His 249) outside of the histidine-rich cluster region was also found to be crucial for activity.

Syringomycin action gene SYR2 is essential for sphingolipid 4-hydroxylation in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae gene SYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis. Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygen-dependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of a syr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids. The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups.

The contryphans, a D-tryptophan-containing family of Conus peptides: interconversion between conformers.

We previously characterized contryphan-R, a D-tryptophan-containing octapeptide from the venom of Conus radiatus. In this study, we present evidence that the contryphan family of peptides is widely distributed in venoms of the fish-hunting cone snails. We purified, synthesized and characterized contryphan-Sm from Conus stercusmuscarum venom, and obtained molecular evidence for the existence of a third peptide, contryphan-P from Conus purpurascens venom ducts.

kappa-Conotoxin PVIIA is a peptide inhibiting the shaker K+ channel.

kappa-Conotoxin PVIIA (kappa-PVIIA), a 27-amino acid toxin from Conus purpurascens venom that inhibits the Shaker potassium channel, was chemically synthesized in a biologically active form. The disulfide connectivity of the peptide was determined. kappa-Conotoxin PVIIA has the following structure.

A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom.

A paralytic peptide, psi-conotoxin Piiie has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of alpha-bungarotoxin, a competitive nAChR antagonist.

MutS mediates heteroduplex loop formation by a translocation mechanism.

Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay.

Strategy for rapid immobilization of prey by a fish-hunting marine snail.

Some venomous animals capture prey with remarkable efficiency and speed. The purple cone, Conus purpurascens, uses two parallel physiological mechanisms requiring multiple neurotoxins to immobilize fish rapidly: neuromuscular block, and excitotoxic shock. The latter requires the newly characterized peptide kappa-conotoxin PVIIA, which inhibits the Shaker potassium channel 2-4, and beta-conotoxin PVIA5, which delays sodium-channel inactivation.

A new family of Conus peptides targeted to the nicotinic acetylcholine receptor.

In this work, a new family of Conus peptides, the alpha A-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, alpha A-conotoxin PIVA, is as follows: [formula: see text] where O represents trans-4-hydroxyproline.

Purification, characterization, synthesis, and cloning of the lockjaw peptide from Conus purpurascens venom.

The major groups of Conus peptides previously characterized from fish-hunting cone snail venoms (the alpha-, mu-, and omega-conotoxins) all blocked neuromuscular transmission. A novel activity, the "lockjaw peptide", from the fish-hunting Conus purpurascens, caused a rigid (instead of flaccid) paralysis in fish and increased excitability at the neuromuscular junction (instead of a block). We report the purification, biological activity, biochemical and preliminary physiological characterization, and chemical synthesis of the lockjaw peptide and the sequence of a cDNA clone encoding its precursor.

Bidirectional excision in methyl-directed mismatch repair.

Using electron microscopy and indirect end-labeling methods, we have examined excision tracts produced by the Escherichia coli methyl-directed mismatch repair system on a closed circular G-T heteroduplex that contains a single d(GATC) site. Despite differing polarities of the unmodified strand in the two hemimethylated derivatives of the heteroduplex, that portion of the unmethylated strand spanning the shorter path between the d(GATC) site and mismatch is targeted for excision in both cases. Mismatch-provoked excision occurring on both hemimethylated DNAs requires DNA helicase II, but exonuclease requirements for the reaction depend on heteroduplex orientation.

Isolation and characterization of the Escherichia coli mutL gene product.

The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone. The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis. The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.

Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis.

A covalently closed, circular heteroduplex containing a G-T mismatch and a single hemimethylated d(GATC) site is subject to efficient methyl-directed mismatch correction in Escherichia coli extracts when repair DNA synthesis is severely restricted by limiting the concentration of exogenously supplied deoxyribonucleoside-5'-triphosphates or by supplementing reactions with chain-terminating 2',3'-dideoxynucleoside triphosphates. However, repair under these conditions results in formation of a single-strand gap in the region of the molecule containing the mismatch and the d(GATC) site. These findings indicate that repair DNA synthesis required for methyl-directed correction can initiate in the vicinity of the mispair, and they are most consistent with a repair reaction involving 3'----5' excision (or strand displacement) from the d(GATC) site followed by 5'----3' repair DNA synthesis initiating in the vicinity of the mismatch.