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

Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P2) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P2, 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. Detailed exploration of the SAR for this novel scaffold resulted in the considerable optimization of both potency for PIP5K, and selectivity over the closely related kinase PI3Kα, as well as identifying several opportunities for the continued optimization of drug-like properties. As a result, several high quality in vitro tool compounds were identified (8, 20 and 25) that demonstrate the desired biochemical and cellular profiles required to aid better understanding of this complex area of biology.

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 1.50 g/cm3), were emplaced within 9-m-deep boreholes, and the first modules were retrieved after 13 months of exposure. Bentonite and associated module materials were sampled, and microbial communities and their distributions were assessed using 16S rRNA gene sequencing and phospholipid fatty acid (PLFA) analysis. Borehole fluid was dominated by amplicon sequence variants (ASVs) affiliated with Desulfosporosinus and Desulfovibrio, which are putatively involved in sulfate reduction. The relative abundance of these ASVs was lower for samples from inside the borehole module, and they were almost undetectable in samples of the inner bentonite layer. The dominant ASV in case and filter sample sequence data was affiliated with Pseudomonas stutzeri, yet its relative abundance decreased in the inner layer samples. Streptomyces sp. ASVs were relatively abundant in all bentonite core sample data both prior to emplacement and after 13 months of exposure, presumably as metabolically inactive spores or extracellular "relic" DNA. PLFA concentrations in outer and inner layer bentonite samples suggested cellular abundances of 1 × 106 to 3 × 106 cells/g, with similar PLFA distributions within all bentonite samples. Our results demonstrate consistent microbial communities inside the saturated borehole module, providing the first evidence for microbial stability under conditions that mimic a deep geological repository.IMPORTANCE The Materials Corrosion Test in Grimsel Underground Research Laboratory, Switzerland, enables an evaluation of microbiological implications of bentonite clay at densities relevant for a deep geological repository. Our research demonstrates that after 13 months of exposure within a granitic host rock, the microbial 16S rRNA gene signatures of saturated bentonite clay within the modules were consistent with the profiles in the original clay used to pack the modules. Such results provide evidence that densities chosen for this emplacement test are refractory to microbial activity, at least on the relatively short time frame leading to the first time point sampling event, which will help inform in situ engineered barrier system science. This study has important implications for the design of deep geological repository sites under consideration for the Canadian Shield.

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 Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination.IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

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 (e.g., 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-1 NaCl, sulfide production varied with temperature, electron donor and bentonite type. Maximum specific sulfide production rates of 0.189 d-1, 0.549 d-1 and 0.157 d-1 occurred in lactate-fed MX-80, Canaprill and National Standard microcosms, respectively. In microcosms with 50 g l-1 NaCl, sulfide production was inhibited. Denaturing gradient gel electrophoresis (DGGE) profiling of microcosms revealed the presence of bacterial classes Clostridia, Bacilli, Gammaproteobacteria, Deltaproteobacteria, Actinobacteria, Sphingobacteriia and Erysipelotrichia. Spore-forming and non-spore-forming bacteria were confirmed in microcosms using high-throughput 16S rRNA gene sequencing. Sulfate-reducing bacteria of the genus Desulfosporosinus predominated in MX-80 microcosms; whereas, Desulfotomaculum and Desulfovibrio genera contributed to sulfate-reduction in National Standard and Canaprill microcosms. CONCLUSIONS: Commercial clays microcosms harbour a sparse bacterial population dominated by spore-forming microorganisms. Detected sulfate- and sulfur-reducing bacteria presumably contributed to sulfide accumulation in the different microcosm systems. SIGNIFICANCE AND IMPACT OF STUDY: The use of carbon-supplemented, clay-in-water microcosms offered insights into the bacterial diversity present in as-received clays, along with the types of metabolic and sulfidogenic reactions that might occur in regions of a DGR (e.g., interfaces between the bulk clay and host rock, cracks, fissures, etc.) that fail to attain target parameters necessary to inhibit microbial growth and activity.

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. As such, understanding transport of sulphide through the engineered barrier system to the used fuel container is an important consideration in DGR design. In this study, a three-dimensional (3D) model of sulphide transport in a DGR was developed. The numerical model is implemented using COMSOL Multiphysics, a commercial finite element software package. Previous sulphide transport models of the NWMO repository used a simplified one-dimensional system. This work illustrates the importance of 3D modelling to capture non-uniform effects, as results showed locations of maximum sulphide flux are 1.7 times higher than the average flux to the used fuel container.

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. The combination of a hygroscopic matrix (i.e., clay or 4,000 MW polyethylene glycol) and high RH resulted in persistent measurable microbial metabolism during desiccation. In contrast, no microbial metabolism was detected at (a) hygroscopic interfaces at low RH, and (b) less hygroscopic interfaces (i.e., sand and plastic/glass) at high or low RH. Cell survival was conversely inhibited at high RH and promoted at low RH, irrespective of surface hygroscopicity. Based on this demonstration of metabolic persistence and survival inhibition at high RH, it was proposed that biofilm metabolic rates might inversely influence whole-biofilm resilience, with 'resilience' defined in this study as a biofilm's capacity to recover from desiccation. The concept of whole-biofilm resilience being promoted by oligotrophy was supported in desiccation-tolerant Arthrobacter spp. biofilms, but not in desiccation-sensitive Pseudomonas aeruginosa biofilms. The ability of microbes to interact with surfaces to harness water vapor during desiccation was demonstrated, and potentially to harness oligotrophy (the most ubiquitous natural condition facing microbes) for adaptation to desiccation.

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. Simple, accessible tools were developed for the study of microbial ecology and metabolic processes that occur within this habitat, since the understanding of the microbiota-niche interaction is fundamental to describing microbial impacts on engineered systems such as compacted bentonite barriers. Even when genomic tools are useful for the study of community composition, techniques to describe such microbial impacts and niche interactions should complement these. Tools optimised for assessing localised microbial activity within bentonite included: (a) the qualitative use of the resazurin-resorufin indicator system for redox localisation, (b) the use of a CaCl2 buffer for the localisation of pH, and (c) fluorometry for the localisation of precipitated sulphide. The use of the Carbon Dioxide Evolution Monitoring System was also validated for measuring microbial activity in desiccated and saturated bentonite. Finally, the buffering of highly-basic bentonite at neutral pH improved the success of isolation of microbial populations, but not DNA, from the bentonite matrix. Thus, accessible techniques were optimised for exploring microbial metabolism in the atypical environments of clay matrices and desiccated conditions. These tools have application to the applied field of used nuclear fuel management, as well as for examining the fundamental biogeochemical cycles active in sedimentary and deep geological environments.

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 ß-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 ß-isoforms with no significant activity towards related kinases in the pathway. In a cellular environment, inhibition of the α- but not the ß-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. Partial least squares analysis identified a metabolic response in the four soils with OC values ≤11% which was well correlated to estimated phenanthrene porewater concentrations. The results suggest that the higher sorption capability of high OC soils decreased the bioavailability of phenanthrene and the subsequent metabolic response of E. fetida.

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.

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). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmols(-1) in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase.

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). Control and contaminant-exposed metabolic profiles were distinguished using principal component analysis and potential contaminant-specific biomarkers of exposure were found for several contaminants. These results suggest that NMR-based metabolomics offers considerable promise for differentiating between the different toxic modes of action (MOA) associated with sub-lethal toxicity to earthworms.

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. Multivariate statistical analysis of 1H NMR spectra for E. fetida tissue extracts indicated that phenanthrene exposed and control earthworms differed throughout the 30 day experiment despite the low phenanthrene concentrations present after 15 days. This metabolic response was better correlated to total phenanthrene concentrations (Q2 = 0.59) than HPCD-extractable phenanthrene concentrations (Q2 = 0.46) suggesting that 1H NMR metabolomics offers considerable promise as a novel, molecular-level method to directly monitor the bioavailability of contaminants to earthworms in the environment.

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. Principal component analysis (PCA) showed differences in responses between exposed earthworms and controls after 48 h exposure. The metabolites that varied with exposure included amino acids (isoleucine, alanine and glutamine) and maltose. PLS models indicated that earthworm response is positively correlated to both total phenanthrene concentration and bioavailable (HPCD-extracted) phenanthrene in a freshly spiked, unaged soil. These results show that metabolomics is a powerful, direct technique that may be used to monitor contaminant bioavailability and toxicity of sub-lethal concentrations of contaminants in the environment. These initial findings warrant further metabolomic studies with aged contaminated soils.

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 0.34 +/- 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R(2)) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5'-CG-3' site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors.

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. Site-specific isotopic variations were large enough to provide proof of principle that quantitative site-specific (2)H NMR may provide an additional parameter for contaminant sourcing at field sites. Isotopic variations were small enough to not bias the comparability of degradation-associated isotopic enrichment factors determined using different MTBE products. Calculated delta(2)H values for MTBE, derived as weighted averages of (2)H NMR measurements of the two functional groups, showed good agreement with IRMS measurements. The ability to gain accurate information about the site-specific isotopic ratios of (2)H/(1)H within a molecule offers considerable promise as a new environmental tool to track the source and fate of environmental contaminants.

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, -1.7 +/- 0.2 per thousand, and -1.7 +/- 0.3 per thousand, respectively. The measured carbon apparent kinetic isotope effect was 1.01 for all compounds, consistent with the expected kinetic isotope effects for both oxidation of the methoxy (or ethoxy) group and enzymatic SN1 biodegradation mechanisms. Significantly, delta13C measurements of the tert-butyl alcohol and tert-amyl alcohol products indicated that the tert-butyl and tert-amyl groups do not participate in the reaction and confirmed that ether biodegradation by strain K1 involves oxidation of the methoxy (or ethoxy) group. Measured hydrogen isotopic enrichment factors (epsilonH) were -100 +/- 10 per thousand, -73 +/- 7 per thousand, and -72 +/- 20 per thousand for MTBE, ETBE, and TAME respectively. Previous results reported for aerobic biodegradation of MTBE by Methylibium petroleiphilum PM1 and Methylibium R8 showed smaller epsilonH values (-35 per thousand and -42 per thousand, respectively). Plots of Delta2H/Delta13C show different slopes for strain K1 compared with strains PM1 and R8, suggesting that different mechanisms are utilized by K1 and PM1/R8 during aerobic MTBE biodegradation.