Identification and optimization of a novel series of selective PIP5K inhibitors.

Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P, PIP5K, has been hypothesized to provide a potential therapeutic target of interest in the treatment of cancers. To better understand and explore the role of PIP5K in human cancers there remains an urgent need for potent and specific PIP5K inhibitor molecules. Following a high throughput screen of the AstraZeneca collection, a novel, moderately potent and selective inhibitor of PIP5K, 1, was discovered.

Stability of Microbial Community Profiles Associated with Compacted Bentonite from the Grimsel Underground Research Laboratory.

To assess the microbiology and corrosion potential of engineered components of a deep geological repository for long-term storage of high-level nuclear waste, the Materials Corrosion Test is being conducted at the Underground Research Laboratory in Grimsel, Switzerland. Modules containing metal coupons surrounded by highly compacted MX-80 bentonite, at two dry densities (1.25 and 1.

Validating DNA Extraction Protocols for Bentonite Clay.

Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material "spiked" with genomic DNA.

Discovery of a Thiadiazole-Pyridazine-Based Allosteric Glutaminase 1 Inhibitor Series That Demonstrates Oral Bioavailability and Activity in Tumor Xenograft Models.

Tumors have evolved a variety of methods to reprogram conventional metabolic pathways to favor their own nutritional needs, including glutaminolysis, the first step of which is the hydrolysis of glutamine to glutamate by the amidohydrolase glutaminase 1 (GLS1). A GLS1 inhibitor could potentially target certain cancers by blocking the tumor cell's ability to produce glutamine-derived nutrients. Starting from the known GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide, we describe the medicinal chemistry evolution of a series from lipophilic inhibitors with suboptimal physicochemical and pharmacokinetic properties to cell potent examples with reduced molecular weight and lipophilicity, leading to compounds with greatly improved oral exposure that demonstrate in vivo target engagement accompanied by activity in relevant disease models.

Bacterial diversity and production of sulfide in microcosms containing uncompacted bentonites.

Aims: This study examined the diversity and sulfide-producing activity of microorganisms in microcosms containing commercial clay products (, MX-80, Canaprill and National Standard) similar to materials which are currently considered for use in the design specifications for deep geologic repositories (DGR) for spent nuclear fuel.

Methods And Results: In anoxic microcosms incubated for minimum of 60 days with 10 g l NaCl, sulfide production varied with temperature, electron donor and bentonite type. Maximum specific sulfide production rates of 0.

Multi-dimensional transport modelling of corrosive agents through a bentonite buffer in a Canadian deep geological repository.

The use of a deep geological repository (DGR) for the long-term disposal of used nuclear fuel is an approach currently being investigated by several agencies worldwide, including Canada's Nuclear Waste Management Organization (NWMO). Within the DGR, used nuclear fuel will be placed in copper-coated steel containers and surrounded by a bentonite clay buffer. While copper is generally thermodynamically stable, corrosion can occur due to the presence of sulphide under anaerobic conditions.

Microbes at Surface-Air Interfaces: The Metabolic Harnessing of Relative Humidity, Surface Hygroscopicity, and Oligotrophy for Resilience.

The human environment is predominantly not aqueous, and microbes are ubiquitous at the surface-air interfaces with which we interact. Yet microbial studies at surface-air interfaces are largely survival-oriented, whilst microbial metabolism has overwhelmingly been investigated from the perspective of liquid saturation. This study explored microbial survival and metabolism under desiccation, particularly the influence of relative humidity (RH), surface hygroscopicity, and nutrient availability on the interchange between these two phenomena.

Measuring microbial metabolism in atypical environments: Bentonite in used nuclear fuel storage.

Genomics enjoys overwhelming popularity in the study of microbial ecology. However, extreme or atypical environments often limit the use of such well-established tools and consequently demand a novel approach. The bentonite clay matrix proposed for use in Deep Geological Repositories for the long-term storage of used nuclear fuel is one such challenging microbial habitat.

Discovery of potent, selective small molecule inhibitors of α-subtype of type III phosphatidylinositol-4-kinase (PI4KIIIα).

The discovery and optimisation of novel, potent and selective small molecule inhibitors of the α-isoform of type III phosphatidylinositol-4-kinase (PI4Kα) are described. Lead compounds show cellular activity consistent with their PI4Kα potency inhibiting the accumulation of IP1 after PDGF stimulation and reducing cellular PIP, PIP2 and PIP3 levels. Hence, these compounds are useful in vitro tools to delineate the complex biological pathways involved in signalling through PI4Kα.

Potent, selective small molecule inhibitors of type III phosphatidylinositol-4-kinase α- but not β-inhibit the phosphatidylinositol signaling cascade and cancer cell proliferation.

Two series of inhibitors of type III phosphatidylinositol-4-kinase were identified by high throughput screening and optimised to derive probe compounds that independently and selectively inhibit the α- and the β-isoforms with no significant activity towards related kinases in the pathway. In a cellular environment, inhibition of the α- but not the β-subtype led to a reduction in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate concentration, causing inhibition of inositol-1-phosphate formation and inhibition of proliferation in a panel of cancer cell lines.

Reduction in the earthworm metabolomic response after phenanthrene exposure in soils with high soil organic carbon content.

We evaluated the correlation between soil organic carbon (OC) content and metabolic responses of Eisenia fetida earthworms after exposure to phenanthrene (58 ± 3 mg/kg) spiked into seven artificial soils with OC contents ranging from 1 to 27% OC. Principal component analysis of (1)H nuclear magnetic resonance (NMR) spectra of aqueous extracts identified statistically significant differences in the metabolic profiles of control and phenanthrene-exposed E. fetida in the 1% OC soil only.

Development of rationally designed DNA N6 adenine methyltransferase inhibitors.

A series of bisubstrate inhibitors for DNA N6 adenine methyltransferase (Dam) have been synthesized by linking an amine analogue of S-adenosylmethionine to an aryl moiety designed to probe the binding pocket of the DNA adenine base. An initial structure-activity relationship study has identified substituents that increase inhibitor potency to the ∼10 μM range and improve selectivity against the human cytosine methyltransferase Dnmt1.

Metabolic responses of Eisenia fetida after sub-lethal exposure to organic contaminants with different toxic modes of action.

Nuclear magnetic resonance (NMR)--based metabolomics has the potential to identify toxic responses of contaminants within a mixture in contaminated soil. This study evaluated the metabolic response of Eisenia fetida after exposure to an array of organic compounds to determine whether contaminant-specific responses could be identified. The compounds investigated in contact tests included: two pesticides (carbaryl and chlorpyrifos), three pharmaceuticals (carbamazephine, estrone and caffeine), two persistent organohalogens (Aroclor 1254 and PBDE 209) and two industrial compounds (nonylphenol and dimethyl phthalate).

Direct and continuous fluorescence-based measurements of Pyrococcus horikoshii DNA N-6 adenine methyltransferase activity.

N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl).

1H NMR metabolomics of earthworm exposure to sub-lethal concentrations of phenanthrene in soil.

1H NMR metabolomics was used to monitor earthworm responses to sub-lethal (50-1500 mg/kg) phenanthrene exposure in soil. Total phenanthrene was analyzed via soxhlet extraction, bioavailable phenanthrene was estimated by hydroxypropyl-beta-cyclodextrin (HPCD) and 1-butanol extractions and sorption to soil was assessed by batch equilibration. Bioavailable phenanthrene (HPCD-extracted) comprised approximately 65-97% of total phenanthrene added to the soil.

Correlations of Eisenia fetida metabolic responses to extractable phenanthrene concentrations through time.

Eisenia fetida earthworms were exposed to phenanthrene for thirty days to compare hydroxypropyl-beta-cyclodextrin (HPCD) extraction of soil and 1H NMR earthworm metabolomics as indicators of bioavailability. The phenanthrene 28-d LC50 value was 750 mg/kg (632-891, 95% confidence intervals) for the peat soil tested. The initial phenanthrene concentration was 319 mg/kg, which biodegraded to 16 mg/kg within 15 days, at which time HPCD extraction suggested that phenanthrene was no longer bioavailable.

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity.

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5'-CG-3' site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.

Quantitative site-specific (2)H NMR investigation of MTBE: potential for assessing contaminant sources and fate.

Site-specific isotopic values of methyl tertiary butyl ether (MTBE) were measured using quantitative site-specific (2)H nuclear magnetic resonance (NMR) spectroscopy for seven commercially available MTBE products. The delta(2)H values of the methoxy and tertiary butyl groups ranged from -103 per thousand to -171 per thousand, and from -76 per thousand to -104 per thousand, reflecting their production from methanol and isobutene, respectively. Several MTBE products whose whole-compound delta(13)C and delta(2)H MTBE values were within error of each other, as measured by isotope ratio mass spectrometry (IRMS), had demonstrably different delta(2)H values for their methoxy and tertiary butyl groups measured by (2)H NMR.

Isotopic fractionation of methyl tert-butyl ether suggests different initial reaction mechanisms during aerobic biodegradation.

Carbon isotopic enrichment factors (epsilonC) measured during cometabolic biodegradation of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by Pseudonocardia tetrahydrofuranoxydans strain K1 were -2.3 +/- 0.2 per thousand, -1.

Phage display screening against a set of targets to establish peptide-based sugar mimetics and molecular docking to predict binding site.

A novel selection approach is presented to screen phage display peptide libraries against sets of receptors that share specificity for the same ligand. This strategy was applied to the discovery of glycomimetic peptides. Through these screens, a number of peptide clones were discovered that bind the lectins used in the screen, in a sugar competitive manner.

Environmental metabolomics: new insights into earthworm ecotoxicity and contaminant bioavailability in soil.

Environmental metabolomics is a growing and emerging sub-discipline of metabolomics. Studies with earthworms have progressed from the initial stages of simple contact exposure tests to detailed studies of earthworm responses in soil. Over the past decade, a variety of endogenous metabolites have been identified as potential biomarkers of contaminant exposure.

Comparative assessments of benzene, toluene, and xylene natural attenuation by quantitative polymerase chain reaction analysis of a catabolic gene, signature metabolites, and compound-specific isotope analysis.

A controlled-release study conducted at Vandenberg Air Force Base involved the injection of anaerobic groundwater amended with benzene, toluene, and o-xylene (BToX; 1-3 mg/L each) in two parallel lanes: lane A injectate contained no ethanol, whereas lane B injectate contained approximately 500 mg/L ethanol. As reported previously by Mackay and co-workers, ethanol led to slower BToX disappearance in lane B. Here, we report on assessments of BToX natural attenuation by three independent and specific monitoring approaches: signature metabolites diagnostic of anaerobic TX metabolism (benzysuccinates), compound-specific isotope analysis (CSIA), and quantitative polymerase chain reaction (qPCR) analysis of a catabolic gene involved in anaerobic TX degradation (bssA).

Insight into methyl tert-butyl ether (MTBE) stable isotope fractionation from abiotic reference experiments.

Methyl group oxidation, SN2-type hydrolysis, and SN1-type hydrolysis are suggested as natural transformation mechanisms of MTBE. This study reports for the first time MTBE isotopic fractionation during acid hydrolysis and for oxidation by permanganate. In acid hydrolysis, MTBE isotopic enrichment factors were epsilon(C) = -4.

Quantifying MTBE biodegradation in the Vandenberg Air Force Base ethanol release study using stable carbon isotopes.

Compound-specific isotope analysis (CSIA) was used to assess biodegradation of MTBE and TBA during an ethanol release study at Vandenberg Air Force Base. Two continuous side-by-side field releases were conducted within a preexisting MTBE plume to form two lanes. The first involved the continuous injection of site groundwater amended with benzene, toluene and o-xylene ("No ethanol lane"), while the other involved the continuous injection of site groundwater amended with benzene, toluene and o-xylene and ethanol ("With ethanol lane").