Discovery of Triterpenoids as Reversible Inhibitors of α/β-hydrolase Domain Containing 12 (ABHD12)

Background

α/β-hydrolase domain containing (ABHD)12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG). Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design.

Methodology/Principal Findings

Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors.

Conclusions/Significance

We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore model of ABHD12 inhibitors. This model should pave the way for further discovery of novel lead structures for ABHD12 selective inhibitors.     

Patterns of Brain Activation when Mothers View Their Own Child and Dog: An fMRI Study

Neural substrates underlying the human-pet relationship are largely unknown. We examined fMRI brain activation patterns as mothers viewed images of their own child and dog and an unfamiliar child and dog. There was a common network of brain regions involved in emotion, reward, affiliation, visual processing and social cognition when mothers viewed images of both their child and dog. Viewing images of their child resulted in brain activity in the midbrain (ventral tegmental area/substantia nigra involved in reward/affiliation), while a more posterior cortical brain activation pattern involving fusiform gyrus (visual processing of faces and social cognition) characterized a mother''s response to her dog. Mothers also rated images of their child and dog as eliciting similar levels of excitement (arousal) and pleasantness (valence), although the difference in the own vs. unfamiliar child comparison was larger than the own vs. unfamiliar dog comparison for arousal. Valence ratings of their dog were also positively correlated with ratings of the attachment to their dog. Although there are similarities in the perceived emotional experience and brain function associated with the mother-child and mother-dog bond, there are also key differences that may reflect variance in the evolutionary course and function of these relationships.     

Influence of elevated ozone and limited nitrogen availability on conifer seedlings in an open-air fumigation system: effects on growth, nutrient content, mycorrhiza, needle ultrastructure, starch and secondary compounds

Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies Karst.) seedlings were exposed to realistically elevated O₃ levels in open‐air experiments over three growing seasons. The total O₃ exposure doses were 1.2 × (1991), 1.5 × (1992) and 1.7 × (1993) ambient levels. During the 1992 and 1993 growing seasons pine and spruce seedlings received two different levels of nitrogen supply. Effects on growth, mycorrhiza formation, needle ultrastructure, primary and secondary compounds were studied. Ozone exposure had only slight effects on biomass production, growth height and nutrient content of studied conifers. Higher nitrogen availability improved growth of the seedlings and resulted in higher concentration of nitrogen in needles. In Scots pine O₃ exposure did not have effects on quantity of total mycorrhizas and short roots, while higher nitrogen availability decreased quantity of mycorrhizas and short roots. In both tree species O₃ exposure induced O₃‐related ultrastructural symptoms, e.g. granulation and dark staining of the chloroplast stroma in the needle mesophyll cells, at both nitrogen availability levels. Ozone exposure and nitrogen availability did not have significant effects on starch concentrations in either tree species. Concentrations of some individual terpenes were higher in O₃‐exposed needles, while concentrations of individual and total resin acids, total phenolics and catechins were not affected by O₃ exposure. Nitrogen availability did not have substantial effects on concentrations of monoterpenes. By contrast, concentrations of some individual and total resin acids were lower in pine needles and higher in spruce needles with higher nitrogen availability, while phenolic concentration in spruce needles decreased at higher nitrogen availability. The results suggest that realistically elevated levels of O₃ in the field can have some negative effects on the mesophyll ultrastructure of conifer needles, but carbon allocation to root and shoot growth and secondary metabolites are not affected substantially.     

Endogenous PttHb1 and PttTrHb, and heterologous Vitreoscilla vhb haemoglobin gene expression in hybrid aspen roots with ectomycorrhizal interaction

Present knowledge on plant non-symbiotic class-1 (Hb1) and truncated (TrHb) haemoglobin genes is almost entirely based on herbaceous species while the corresponding tree haemoglobin genes are not well known. The function of these genes has recently been linked with endosymbioses between plants and microbes. In this work, the coding sequences of hybrid aspen (Populus tremulaxtremuloides) PttHb1 and PttTrHb were characterized, indicating that the key residues of haem and ligand binding of both genes were conserved in the deduced amino acid sequences. The expression of PttHb1 and PttTrHb was examined in parallel with that of the heterologous Vitreoscilla haemoglobin gene (vhb) during ectomycorrhiza/ectomycorrhizal (ECM) interaction. Both ECM fungi studied, Leccinum populinum and Xerocomus subtomentosus, enhanced root formation and subsequent growth of roots of all hybrid aspen lines, but only L. populinum was able to form mycorrhizas. Real-time PCR results show that the dual culture with the ECM fungus, with or without emergence of symbiotic structures, increased the expression of both PttHb1 and PttTrHb in the roots of non-transgenic hybrid aspens. PttHb1 and PttTrHb had expression peaks 5 h and 2 d after inoculation, respectively, pointing to different functions for these genes during interaction with root growth-improving fungi. In contrast, ECM fungi were not able to enhance the expression of hybrid aspen endogenous haemoglobin genes in the VHb lines, which may be a consequence of the compensating action of heterologous haemoglobin.     

Cloning and expression in a heterologous host of the complete set of genes for biosynthesis of the Streptomyces coelicolor antibiotic undecylprodigiosin

A fragment of DNA carrying the hitherto unisolated members of the cluster of genes (red) for biosynthesis of the red-pigmented antibiotic undecylprodigiosin of Streptomyces coelicolor A3(2) was isolated. This was done by cloning random fragments of S. coelicolor DNA into the closely related Streptomyces lividans 66 and recovering a clone that caused overproduction of undecylprodigiosin. The effect was probably due to the presence of the cloned redD gene, which functions as a positive regulator of the expression of the red cluster, activating the normally poorly expressed red genes of S. lividans. Two fragments from either end of the red cluster were cloned adjacent to each other on a low-copy-number Streptomyces vector. Double crossing-over occurring between these plasmid-borne sequences and the chromosomal copy of the same DNA in S. coelicolor led to isolation of the entire red cluster as a single cloned fragment. Isolation of antibiotic biosynthetic genes by the effects of an activator in a self-cloning experiment, and in vivo reconstitution of a large cluster of genes by homologous recombination, may turn out to be usefully generalizable procedures.     

Effects of drought and waterlogging on ultrastructure of Scots pine and Norway spruce needles

Effects of water stress on needle ultrastructure of 2-year-old Scots pine (Pinus sylvestris L.) and 5-year-old Norway spruce [Picea abies (L.) Karst.] seedlings were studied in greenhouse experiments. Drought stress was induced by leaving seedlings without watering, and waterlogging stress was produced by submerging the seedling containers in water. Needle samples for ultrastructural analyses were collected several times during the experiments, and samples for nutrient analyses at the end of the experiments. In drought stress, plasmolysis of mesophyll and transfusion parenchyma tissues, aggregation of chloroplast stroma and its separation from thylakoids and decreased size and abundance of starch grains in needles of both species were observed. The concentration of lipid bodies around the chloroplasts were detected in pine needles. Calcium and water concentrations in spruce needles were lower by the end of the experiments compared to controls. In waterlogging treatment, swelling of phloem cells in pine needles and large starch grains, slight swelling of thylakoids and increased translucency of plastoglobuli in chloroplasts of both species studied were observed. The phosphorus concentration in pine needles was higher while phosphorus, calcium and magnesium concentrations in spruce needles were lower in the waterlogging treatments compared to controls. Typical symptoms induced by drought stress, e. g. aggregation of chloroplast stroma and its separation from thylakoids, were detected, but, in waterlogging stress, ultrastructural symptoms appeared to be related to the developing nutrient imbalance of needles.     

Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia

Application of DNA fingerprinting methods enables the detection of diverse members of soil bacterial consortia, even including those bacteria not yet cultivated. However, extraction and purification of DNA from soil samples without bias is difficult. We compared five different DNA isolation methods and three purification methods for rhizosphere soil samples. Purified DNA extracts were amplified in PCR using universal bacterial primers and the PCR products were analysed with denaturing gradient gel electrophoresis (DGGE) for the visualisation of DNA bands representing dominant bacterial species. Both the isolation and purification methods affected the apparent bacterial community structure of the samples.     

A new approach to high sensitivity differential hybridization

We describe a new approach to differential hybridization, designed to identify cDNA clones representing rare mRNA species. Duplicate filters carrying a library of cDNA from phorbolmyristate acetate (PMA)-induced EL-4 cells in λgt11 were hybridized with high concentrations of unlabeled, cloned, single-stranded cDNA from induced and control EL-4 cells, respectively. Plaques binding single-stranded cDNA were revealed by a second round of hybridization with ³⁵S-labeled DNA complementary to the vector moiety of the single-stranded cDNA. Plaques corresponding to PMA-induced mRNAs occurring at a level of about 1 part in 15000 were isolated. We believe the method is at least ten times more sensitive than conventional differential hybridization.