Activation of Adenosine Monophosphate-Activated Protein Kinase Reduces the Onset of Diet-Induced Hepatocellular Carcinoma in Mice.

The worldwide obesity and type 2 diabetes epidemics have led to an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD covers a spectrum of hepatic pathologies ranging from simple steatosis to nonalcoholic steatohepatitis, characterized by fibrosis and hepatic inflammation. Nonalcoholic steatohepatitis predisposes to the onset of hepatocellular carcinoma (HCC). Here, we characterized the effect of a pharmacological activator of the intracellular energy sensor adenosine monophosphate-activated protein kinase (AMPK) on NAFLD progression in a mouse model. The compound stimulated fat oxidation by activating AMPK in both liver and skeletal muscle, as revealed by indirect calorimetry. This translated into an ameliorated hepatic steatosis and reduced fibrosis progression in mice fed a diet high in fat, cholesterol, and fructose for 20 weeks. Feeding mice this diet for 80 weeks caused the onset of HCC. The administration of the AMPK activator for 12 weeks significantly reduced tumor incidence and size. Conclusion: Pharmacological activation of AMPK reduces NAFLD progression to HCC in preclinical models.

Synthesis of Shld Derivatives, Their Binding to the Destabilizing Domain, and Influence on Protein Accumulation in Transgenic Plants.

A series of 35 analogues of Shld with modifications in the A-residue and the C-residues were prepared and investigated for binding to FKBP and GFP accumulation in transgenic plants. The modifications investigated explored variations that were supposedly inside or outside the receptor binding site with the latter being important by influencing the overall polarity of the compounds in order to improve the absorption in plants. The binding of the new compounds to the destabilizing domain was determined using a fluorescence polarization competition assay, and the GFP expression in engineered Arabidopsis thaliana was studied. The results showed that modifications of the C-building block phenol with acidic, basic, and neutral groups led to better ligands with some being better than Shld in the plant. Generally small, polar substituents showed the best GFP accumulation.

Determination of protonation states of iminosugar-enzyme complexes using photoinduced electron transfer.

A series of N-alkylated analogues of 1-deoxynojirimycin containing a fluorescent 10-chloro-9-anthracene group in the N-alkyl substituent were prepared. The anthracene group acted as a reporting group for protonation at the nitrogen in the iminosugar because an unprotonated amine was found to quench fluorescence by photoinduced electron transfer. The new compounds were found to inhibit ß-glucosidase from Phanerochaete chrysosporium and α-glucosidase from Aspergillus niger, with Ki values in the low micro- to nanomolar range. Fluorescence and inhibition versus pH studies of the ß-glucosidase-iminosugar complexes revealed that the amino group in the inhibitor is unprotonated when bound, while one of the active site carboxylates is protonated.

Selenoureido-iminosugars: A new family of multitarget drugs.

Herein we report the synthesis of N-alkylated deoxynojirimycin derivatives decorated with a selenoureido motif at the hydrocarbon tether as an example of unprecedented multitarget agents. Title compounds were designed as dual drugs for tackling simultaneously the Gaucher disease (by selective inhibition of ß-glucosidase, Ki = 1.6-5.5 µM, with improved potency and selectivity compared to deoxynojirimycin) and its neurological complications (by inhibiting AChE, Ki up to 5.8 µM). Moreover, an excellent mimicry of the selenoenzyme glutathione peroxidase was also found for the catalytic scavenging of H2O2 (Kcat/Kuncat up to 640) using PhSH as a cofactor, with improved activity compared to known positive controls, like (PhSe)2 and ebselen; therefore, such compounds are also excellent scavengers of peroxides, an example of reactive oxygen species present at high concentrations in patients of Gaucher disease and neurological disorders.

Intraspecific variation of a dominant grass and local adaptation in reciprocal garden communities along a US Great Plains' precipitation gradient: implications for grassland restoration with climate change.

Identifying suitable genetic stock for restoration often employs a 'best guess' approach. Without adaptive variation studies, restoration may be misguided. We test the extent to which climate in central US grasslands exerts selection pressure on a foundation grass big bluestem (Andropogon gerardii), widely used in restorations, and resulting in local adaptation. We seeded three regional ecotypes of A. gerardii in reciprocal transplant garden communities across 1150 km precipitation gradient. We measured ecological responses over several timescales (instantaneous gas exchange, medium-term chlorophyll absorbance, and long-term responses of establishment and cover) in response to climate and biotic factors and tested if ecotypes could expand range. The ecotype from the driest region exhibited greatest cover under low rainfall, suggesting local adaptation under abiotic stress. Unexpectedly, no evidence for cover differences between ecotypes exists at mesic sites where establishment and cover of all ecotypes were low, perhaps due to strong biotic pressures. Expression of adaptive differences is strongly environment specific. Given observed adaptive variation, the most conservative restoration strategy would be to plant the local ecotype, especially in drier locations. With superior performance of the most xeric ecotype under dry conditions and predicted drought, this ecotype may migrate eastward, naturally or with assistance in restorations.

Exploring the relationship between the conformation and pKa: can a pKa value be used to determine the conformational equilibrium?

Four substituted cis and trans-4,5-dihydroxyhexahydropyridazines that were expected to undergo pH induced conformational switching were synthesized and carefully investigated by NMR analyses and calculations. For two of the compounds a large difference in pKa existed between the two possible chair conformers and for one compound this resulted in conformational switching as a result of pH change. For the first time it is shown that the pKa directly reflects the conformational equilibrium of conformers.

Design, synthesis and in vitro pharmacology of GluK1 and GluK3 antagonists. Studies towards the design of subtype-selective antagonists through 2-carboxyethyl-phenylalanines with substituents interacting with non-conserved residues in the GluK binding sites.

In order to identify compounds selective for the GluK1 and GluK3 subtypes of kainate receptors we have designed and synthesized a series of (S)-2-amino-3-((2-carboxyethyl)phenyl)propanoic acid analogs with hydrogen bond donating and accepting substituents on the aromatic ring. Based on crystal structures of GluK1 in complex with related ligands, the compounds were designed to explore possible interactions with non-conserved residues outside the glutamate ligand binding site and challenge the water binding network. Apart from obtaining GluK1 selective antagonists one analog with a phenyl-substituted urea (compound 31) showed some preference for GluK3 over GluK1-receptors. Docking studies indicate that this preference may be attributed to contacts between the NH of the urea substituent and non-conserved Ser741 and Ser761 residues.

Environmental and genetic variation in leaf anatomy among populations of Andropogon gerardii (Poaceae) along a precipitation gradient.

PREMISE OF THE STUDY: Phenotypes of two Andropogon gerardii subspecies, big bluestem and sand bluestem, vary throughout the prairie ecosystem of North America. This study sought to determine the role of genetics and environment in driving adaptive variation of leaf structure in big bluestem and sand bluestem. • METHODS: Four populations of big bluestem and one population of sand bluestem were planted in common gardens at four sites across a precipitation gradient from western Kansas to southern Illinois. Internal leaf structure and trichome density of A. gerardii were examined by light microscopy to separate genetic and environmentally controlled traits. Leaf thickness, midrib thickness, bulliform cells, interveinal distance, vein size, and trichome density were quantified. • KEY RESULTS: At all planting sites, sand bluestem and the xeric population of A. gerardii had thicker leaves and fewer bulliform cells compared with mesic populations. Environment and genetic source population were both influential for leaf anatomy. Leaves from plants grown in mesic sites (Carbondale, Illinois and Manhattan, Kansas) had thicker midribs, larger veins, fewer trichomes, and a greater proportion of bulliform cells compared to plants grown in drier sites (Colby and Hays, Kansas). • CONCLUSIONS: Water availability has driven adaptive variation in leaf structure in populations of A. gerardii, particularly between sand bluestem and big bluestem. Genetically based differences in leaves of A. gerardii indicate adaptive variation and evolutionary forces differentiating sand bluestem from big bluestem. Environmental responses of A. gerardii leaves suggest an ability to adjust to drought, even in populations adapted to mesic home environments.

Eye movement suppression interferes with construction of object-centered spatial reference frames in working memory.

The brain's frontal eye fields (FEF), responsible for eye movement control, are known to be involved in spatial working memory (WM). In a previous fMRI experiment (Wallentin, Roepstorff & Burgess, Neuropsychologia, 2008) it was found that FEF activation was primarily related to the formation of an object-centered, rather than egocentric, spatial reference frame. In this behavioral experiment we wanted to demonstrate a causal relationship between eye movement control and manipulation of spatial reference frames. Sixty-two participants recalled either spatial ("Was X in front of Y?") or non-spatial ("Was X darker than Y?") relations in a previously shown image containing two to four objects, each with an intrinsic orientation and unique luminance. During half of all recall trials a moving visual stimulus was presented, which participants had to ignore, thus suppressing eye movement. Response times were significantly slower for spatial relations with distraction while there was no effect on non-spatial relations. There was no effect on accuracy, i.e. WM maintenance. This is consistent with the hypothesis that in spatial representations the FEFs are involved in WM content manipulation, such as establishing an object-centered spatial frame of reference.

Quantifying the electronic effects of carbohydrate hydroxy groups by using aminosugar models.

Methyl amino-deoxy-glycosides with α- and ß-gluco, α-galacto, or α-manno stereochemistry with the amino functionality in each of the four possible non-anomeric positions have been synthesized and their pK(a) values determined by titration. These model compounds were chosen because they are the amino derivatives of the most common glycosyl acceptors. From this study it was possible to evaluate the electron density at each of the given positions in the carbohydrate and compare them. Some general trends were observed: The basicity of the amino groups decreases in the order 6-NH(2)>3-NH(2)>2-NH(2)>4-NH(2) (referring to the position). The basicity of a of an amino-deoxy-sugar generally increases when one or more substituents on the sugar ring are axial. The basicity decreases when the amine is antiperiplanar to an oxygen atom. These findings are in agreement with the observations obtained from glycosylation chemistry and the regioselective protection of sugars.

A fluorine scan at the catalytic center of thrombin: C--F, C--OH, and C--OMe bioisosterism and fluorine effects on pKa and log D values.

A series of 16 tricyclic thrombin inhibitors was prepared by using the 1,3-dipolar cycloaddition of azomethine ylides derived from 3- or 4-hydroxyproline and 4-bromobenzaldehyde, with N-(4-fluorobenzyl)maleimide as the key step. The terminal pyrrolidine ring of the inhibitors was systematically substituted to explore the potential bioisosteric behavior of C-F, C-OH, and C-OMe residues pointing into the environment of the catalytic center of a serine protease. X-ray crystal structure analyses revealed a distinct puckering preference of this ring. Substitution by F, HO, and MeO has a strong effect on the basicity of the adjacent pyrrolidine nitrogen center which originates from two sigma-inductive pathways between this center and the electronegative O and F atoms. gem-Difluorination decreases the pKa value of this tertiary amine center to <2, making the conjugated ammonium ion a moderately strong acid. Unexpectedly, F substitution next to the nitrogen center reduced the lipophilicity of the ligands, as revealed by measurements of the logarithmic partition coefficient log D. The biological assays showed that all compounds are thrombin inhibitors with activities between Ki=0.08 and 2.17 microM. Bioisosteric behavior of F, HO, and MeO substituents was observed. Their electronegative F and O atoms undergo energetically similar polar interactions with positively polarized centers, such as the N atom of His 57 which is hydrogen bonded to the catalytic Ser 195. However, for energetically similar polar interactions of C-F, C-OH, and C-OMe to occur, sufficient space is necessary for the accommodation of the Me group of the C-OMe residue, and a H-bond acceptor must be present to prevent unfavorable desolvation of the C-OH residue.

Multipolar interactions in the D pocket of thrombin: large differences between tricyclic imide and lactam inhibitors.

Two series of tricyclic inhibitors of the serine protease thrombin, imides (+/-)-1-(+/-)-8 and lactams (+/-)-9-(+/-)-13, were analysed to evaluate contributions of orthogonal multipolar interactions with the backbone C=O moiety of Asn98 to the free enthalpy of protein-ligand complexation. The lactam derivatives are much more potent and more selective inhibitors (K(i) values between 0.065 and 0.005 microM, selectivity for thrombin over trypsin between 361- and 1609-fold) than the imide compounds (Ki values between 0.057 and 23.7 microM, selectivity for thrombin over trypsin between 3- and 67-fold). The increase in potency and selectivity is explained by the favorable occupancy of the P-pocket of thrombin by the additional isopropyl substituent in the lactam derivatives. The nature of the substituent on the benzyl ring filling the D pocket strongly influences binding potency in the imide series, with Ki values increasing in the sequence: F < OCH2O < Cl < H < OMe < OH < N(pyr)<< Br. This sequence can be explained by both steric fit and the occurrence of orthogonal multipolar interactions with the backbone C[double bond, length as m-dash]O moiety of Asn98. In contrast, the substituent on the benzyl ring hardly affects the ligand potency in the lactam series. This discrepancy was clarified by the comparison of X-ray structures solved for co-crystals of thrombin with imide and lactam ligands. Whereas the benzyl substituents in the imide inhibitors are sufficiently close (< or =3.5 Angstroms) to the C=O group of Asn98 to allow for attractive orthogonal multipolar interactions, the distances in the lactam series are too large (> or =4 Angstroms) for attractive dipolar contacts to be effective.

Fluorine interactions at the thrombin active site: protein backbone fragments H-C(alpha)-C=O comprise a favorable C-F environment and interactions of C-F with electrophiles.

In a systematic fluorine scan of a rigid inhibitor to map the fluorophilicity/fluorophobicity of the active site in thrombin, one or more F substituents were introduced into the benzyl ring reaching into the D pocket. The 4-fluorobenzyl inhibitor showed a five to tenfold higher affinity than ligands with other fluorination patterns. X-ray crystal-structure analysis of the protein-ligand complex revealed favorable C-F...H-C(alpha)-C=O and C-F...C=O interactions of the 4-F substituent of the inhibitor with the backbone H-C(alpha)-C=O unit of Asn98. The importance of these interactions was further corroborated by the analysis of small-molecule X-ray crystal-structure searches in the Protein Data Base (PDB) and the Cambridge Structural Database (CSD). In the C--F...C=O interactions that are observed for both aromatic and aliphatic C-F units and a variety of carbonyl and carboxyl derivatives, the F atom approaches the C=O C atom preferentially along the pseudotrigonal axis of the carbonyl system. Similar orientational preferences are also seen in the dipolar interactions C--F.C[triple chemical bond]N, C-F.C-F, and C-F...NO(2), in which the F atoms interact at sub-van der Waals distances with the electrophilic centers.

A fluorine scan of the phenylamidinium needle of tricyclic thrombin inhibitors: effects of fluorine substitution on pKa and binding affinity and evidence for intermolecular C-F...CN interactions.

The H-atoms of the phenylamidinium needle of tricyclic thrombin inhibitors, which interacts with Asp189 at the bottom of the selectivity pocket S1 of the enzyme, were systematically exchanged with F-atoms in an attempt to improve the pharmacokinetic properties by lowering the pK(a) value. Both the pK(a) values and the inhibitory constants K(i) against thrombin and trypsin were decreased upon F-substitution. Interestingly, linear free energy relationships (LFERs) revealed that binding affinity against thrombin is much more affected by a decrease in pK(a) than the affinity against trypsin. Surprising effects of F-substitutions in the phenylamidinium needle on the pK(a) value of the tertiary amine centre in the tricyclic scaffold of the inhibitors were observed and subsequently rationalised by X-ray crystallographic analysis and ab initio calculations. Evidence for highly directional intermolecular C-F...CN interactions was obtained by analysis of small-molecule X-ray crystal structures and investigations in the Cambridge Structural Database (CSD).

Identification of potent and broad-spectrum antibiotics from SAR studies of a synthetic vancomycin analogue.

Dimeric vancomycin analogues based on a lead compound identified from a library of synthetic analogues of vancomycin have up to 60-fold greater activity than vancomycin against vancomycin-resistant Enterococcus faecium (VRE, VanA phenotype). Simplified analogues have also been prepared and found to maintain activity against VRE and have broad-spectrum antibiotic activity.