Spectrophotometric determination of α-hydroxytropolone pK a values: A structure-acidity relationship study.

α-Hydroxytropolones (αHTs) have a wealth of biological activity owing to their ability to serve as metalbinding fragments for many therapeutically valuable dinuclear metalloenzymes. They also have the potential to exist in as many as 4 protonation states under aqueous acidic or basic conditions. The following details how UV absorption can be used to generate pK a values on a series of αHTs. The studies also provide some knowledge into how the acidity and basicity change with some different functional groups. These studies thus provide new strategies and knowledge that could be valuable in leveraging αHTs as metal-binding fragments in drug-development pursuits.

Troponoids Can Inhibit Growth of the Human Fungal Pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a pathogen that is common in immunosuppressed patients. It can be treated with amphotericin B and fluconazole, but the mortality rate remains 15 to 30%. Thus, novel and more effective anticryptococcal therapies are needed. The troponoids are based on natural products isolated from western red cedar, and have a broad range of antimicrobial activities. Extracts of western red cedar inhibit the growth of several fungal species, but neither western red cedar extracts nor troponoid derivatives have been tested against C. neoformans We screened 56 troponoids for their ability to inhibit C. neoformans growth and to assess whether they may be attractive candidates for development into anticryptococcal drugs. We determined MICs at which the compounds inhibited 80% of cryptococcal growth relative to vehicle-treated controls and identified 12 compounds with MICs ranging from 0.2 to 15 µM. We screened compounds with MICs of ≤20 µM for cytotoxicity in liver hepatoma cells. Fifty percent cytotoxicity values (CC50s) ranged from 4 to >100 µM. The therapeutic indexes (TI, CC50/MIC) for most of the troponoids were fairly low, with most being <8. However, two compounds had TI values that were >8, including a tropone with a TI of >300. These tropones are fungicidal and are not antagonistic when used in combination with fluconazole or amphotericin B. Inhibition by these two tropones remains unchanged under conditions favoring cryptococcal capsule formation. These data support the hypothesis that troponoids may be a productive scaffold for the development of novel anticryptococcal therapies.

Efficacy of hepatitis B virus ribonuclease H inhibitors, a new class of replication antagonists, in FRG human liver chimeric mice.

Chronic hepatitis B virus infection cannot be cured by current therapies, so new treatments are urgently needed. We recently identified novel inhibitors of the hepatitis B virus ribonuclease H that suppress viral replication in cell culture. Here, we employed immunodeficient FRG KO mice whose livers had been engrafted with primary human hepatocytes to ask whether ribonuclease H inhibitors can suppress hepatitis B virus replication in vivo. Humanized FRG KO mice infected with hepatitis B virus were treated for two weeks with the ribonuclease H inhibitors #110, an α-hydroxytropolone, and #208, an N-hydroxypyridinedione. Hepatitis B virus viral titers and S and e antigen plasma levels were measured. Treatment with #110 and #208 caused significant reductions in plasma viremia without affecting hepatitis B virus S or e antigen levels, and viral titers rebounded following treatment cessation. This is the expected pattern for inhibitors of viral DNA synthesis. Compound #208 suppressed viral titers of both hepatitis B virus genotype A and C isolates. These data indicate that Hepatitis B virus replication can be suppressed during infection in an animal by inhibiting the viral ribonuclease H, validating the ribonuclease H as a novel target for antiviral drug development.

PHOXI: A High Quantum Yield, Solvent-Sensitive Blue Fluorescent 5-Hydroxytryptophan Derivative Synthesized within Ten Minutes under Aqueous, Ambient Conditions.

Multiple tryptophan (Trp) proteins are not amenable to fluorescence study because individual residue emission is not resolvable. Biosynthetic incorporation of an indole analogue such as 5-hydroxyindole has not provided sufficient spectroscopic resolution because of low quantum yield and small emission shift. Here, 5-hydroxyindole is used as the starting framework for building a blue emitting fluorophore of high quantum yield, 2-phenyl-6H-oxazolo[4,5-e]indole (PHOXI). This is a three reagent reaction completed in 10 min under ambient conditions in borate buffer at pH 8. Reaction conditions have been optimized using 5-hydroxyindole. Derivatization is demonstrated on tryptophanyl 5-hydroxytryptophan (5-HTP) and a stable ß-hairpin "zipper" peptide with four tryptophan residues, TrpZip2, where Trp 4 has been replaced with 5-HTP, W4 → 5-HTP. Reaction optimization yields a PHOXI fluorophore that is essentially free of byproducts. Reaction specificity is demonstrated by the lack of reaction with N-acetyl-cysteine and amyloid ß-40, a peptide containing all amino acids except tryptophan, proline, and cysteine and lacking 5-HTP. Fluorescence study of PHOXI-derivatized 5-hydroxyindole in different solvents reveals the sensitivity of PHOXI to solvent polarity with a remarkable 87 nm red-shift in water relative to cyclohexane while maintaining high quantum yield. Thus, PHOXI joins the ranks of solvatochromic fluorophores such as PRODAN. Surprisingly, DFT calculations reveal coplanarity of the oxazolo/indole extended ring system and the phenyl substituent for both the HOMO and LUMO orbitals. Despite the crowded environment of three additional Trps in TrpZip2, CD spectroscopy shows that the TrpZip2 ß-hairpin structure is partially retained upon PHOXI incorporation. In an environment of smaller residues, PHOXI incorporation can be less disruptive of protein secondary structure, especially at molecular interfaces and other environments where there is typically less steric hindrance.

Sample limited characterization of a novel disulfide-rich venom peptide toxin from terebrid marine snail Terebra variegata.

Disulfide-rich peptide toxins found in the secretions of venomous organisms such as snakes, spiders, scorpions, leeches, and marine snails are highly efficient and effective tools for novel therapeutic drug development. Venom peptide toxins have been used extensively to characterize ion channels in the nervous system and platelet aggregation in haemostatic systems. A significant hurdle in characterizing disulfide-rich peptide toxins from venomous animals is obtaining significant quantities needed for sequence and structural analyses. Presented here is a strategy for the structural characterization of venom peptide toxins from sample limited (4 ng) specimens via direct mass spectrometry sequencing, chemical synthesis and NMR structure elucidation. Using this integrated approach, venom peptide Tv1 from Terebra variegata was discovered. Tv1 displays a unique fold not witnessed in prior snail neuropeptides. The novel structural features found for Tv1 suggest that the terebrid pool of peptide toxins may target different neuronal agents with varying specificities compared to previously characterized snail neuropeptides.

Geobacillus gargensis sp. nov., a novel thermophile from a hot spring, and the reclassification of Bacillus vulcani as Geobacillus vulcani comb. nov.

A novel thermophilic spore-forming strain, Ga(T), was isolated from the Garga hot spring located in the northern part of the Transbaikal region (Russia). Strain Ga(T) was found to be an aerobic, Gram-positive, rod-shaped, thermophilic (optimum growth temperature is 60-65 degrees C), chemo-organotrophic bacterium that grows on various sugars, carboxylic acids and hydrocarbons. The G+C content of its DNA is 52.9 mol%. The 16S rRNA gene sequence similarity data show that strain Ga(T) is closely related to members of the genus Geobacillus. Relevant chemotaxonomic data (in particular, the major fatty acid profile of strain Ga(T), which includes iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0 acids) support the assignment of this strain to the genus Geobacillus. The physiological, biochemical and DNA-DNA hybridization studies of strain Ga(T) showed that it differs both genotypically and phenotypically from the recognized Geobacillus species. Based on these data, strain Ga(T) belongs to a novel species, Geobacillus gargensis sp. nov. (type strain, Ga(T)=VKM B-2300(T)=DSM 15378(T)). The analysis of the phenotypic characteristics (additional to those given in the original description) of the type strain of Bacillus vulcani (DSM 13174(T)) showed that they are very similar to the major phenotypic characteristics of the genus Geobacillus. The low DNA-DNA reassociation values of strain DSM 13174(T) with various species of this genus (from 38 to 54 %) clearly demonstrate a sufficient genomic distinction of this strain and its taxonomic status as a species. The physiological characteristics, phylogenetic position and DNA-DNA reassociation values of B. vulcani allow this species to be reclassified as Geobacillus vulcani comb. nov. The main properties that differentiate G. vulcani from the other species of the genus are its ability to produce acids from glycerol, lactose and ribose.