AMPK/α-Ketoglutarate Axis Regulates Intestinal Water and Ion Homeostasis in Young Pigs.

Water and ion absorption via sensitive aquaporins (AQPs) and ion channels is of critical importance in intestinal health. However, whether α-ketoglutarate (AKG) could improve intestinal water and ion homeostasis in lipopolysaccharide (LPS)-challenged piglets and whether the AMP-activated protein kinase (AMPK) pathway is involved remains largely unknown. This study was conducted to investigate the effect of dietary AKG supplementation on the small intestinal water and ion homeostasis through modulating the AMPK pathway in a piglet diarrhea model. A total of 32 weaned piglets were used in a 2 × 2 factorial design; the major factors were diet (basal diet or 1% AKG diet) and challenge (Escherichia coli LPS or saline). The results showed that LPS challenge increased the diarrhea index and affected the concentrations of serum Na+, K+, Cl-, glucose, and AKG and its metabolites in piglets fed the basal or AKG diet. However, the addition of AKG attenuated diarrhea incidence and reversed these serum parameter concentrations. Most AQPs (e.g., AQP1, AQP3, AQP4, AQP5, AQP8, AQP10, and AQP11) and ion transporters (NHE3, ENaC, and DRA/PAT1) were widely distributed in the duodenum and jejunum of piglets. We also found that AKG up-regulated the expression of intestinal epithelial AQPs while inhibiting the expression of ion transporters. LPS challenge decreased (P < 0.05) the gene and protein expression of the AMPK pathway (AMPKα1, AMPKα2, SIRT1, PGC-1α, ACC, and TORC2) in the jejunum and ileum. Notably, AKG supplementation enhanced the abundance of these proteins in the LPS-challenged piglets. Collectively, AKG plays an important role in increasing water and ion homeostasis through modulating the AMPK pathway. Our novel finding has important implications for the prevention and treatment of gut dysfunction in neonates.

Effects of Alpha-Ketoglutarate on Glutamine Metabolism in Piglet Enterocytes in Vivo and in Vitro.

Alpha-ketoglutarate (AKG) plays a vital part in the tricarboxylic acid cycle and is a key intermediate in the oxidation of L-glutamine (Gln). The study was to evaluate effects of AKG on Gln metabolism in vivo and in vitro. A total of twenty-one piglets were weaned at 28 days with a mean body weight (BW) of 6.0 ± 0.2 kg, and randomly divided into 3 groups: corn soybean meal based diet (CON group); the basal diet with 1% alpha-ketoglutarate (AKG treatment group); and the basal diet with 1% L-glutamine (GLN treatment group). Intestinal porcine epithelial cells-1 (IPEC-1) were incubated to investigate effects of 0.5, 2, and 3 mM AKG addition on Gln metabolism. Our results showed that there were no differences (P > 0.05) among the 3 treatments in initial BW, final BW, and average daily feed intake. However, average daily gain (P = 0.013) and gain:feed (P = 0.041) of the AKG group were greater than those of the other two groups. In comparison with the CON group, the AKG and GLN groups exhibited an improvement in villus length, mucosal thickness, and crypt depth in the jejunum of piglets. Serum concentrations of Asp, Glu, Val, Ile, Tyr, Phe, Lys, and Arg in the piglets fed the 1% AKG or Gln diet were lower than those in the CON group. Compared with the CON group, the mRNA expression of jejunal and ileal amino acid (AA) transporters in the AKG and GLN groups were significantly increased (P < 0.05). Additionally, the in vitro study showed that the addition of 0.5, 2, and 3 mM AKG dose-dependently decreased (P < 0.05) the net utilization of Gln and formulation of ammonia, Glu, Ala, and Asp by IPEC-1. In conclusion, dietary AKG supplementation, as a replacement for Gln, could improve Gln metabolism in piglet enterocytes and enhance the utilization of AA.

Structure-Based Discovery of Novel and Selective 5-Hydroxytryptamine 2B Receptor Antagonists for the Treatment of Irritable Bowel Syndrome.

Here we employed structure-based ligand discovery techniques to explore a recently determined crystal structure of the 5-hydroxytryptamine 2B (5-HT2B) receptor. Ten compounds containing a novel chemical scaffold were identified; among them, seven molecules were active in cellular function assays with the most potent one exhibiting an IC50 value of 27.3 nM. We then systematically probed the binding characteristics of this scaffold by designing, synthesizing, and testing a series of structural modifications. The structure-activity relationship studies strongly support our predicted binding model. The binding profiling across a panel of 11 5-HT receptors indicated that these compounds are highly selective for the 5-HT2B receptor. Oral administration of compound 15 (30 mg/kg) produced significant attenuation of visceral hypersensitivity in a rat model of irritable bowel syndrome (IBS). We expect this novel scaffold will serve as the foundation for the development of 5-HT2B antagonists for the treatment of IBS.

Evaluation and application of MD-PB/SA in structure-based hierarchical virtual screening.

Molecular dynamics (MD) based molecular mechanics Poisson-Boltzmann and surface area (MM-PB/SA) calculation (MD-PB/SA) has been widely used to estimate binding free energies for receptor-ligand complexes. While numerous reports have focused on assessing accuracy and efficiency, fewer studies have paid attention to performance in lead discovery. In the present study, we report a critical evaluation of MD-PB/SA in hierarchical virtual screening (HVS) both theoretically and practically. It is shown that based on native poses, MD-PB/SA could be well applied to predict the relative binding energy for both congeneric and diverse ligands for different protein targets. However, there is a limitation for MD-PB/SA to distinguish the native pose of one ligand from the artificial pose of another when a huge difference exists between two molecules. By combining a physics-based scoring function with a knowledge-based structural filter, we improve the predictability and validate the practical use of MD-PB/SA in lead discovery by identifying novel inhibitors of p38 MAP kinase. We also expand our study to other protein targets such as HIV-1 RT and NA to assess the general validity of MD-PB/SA.

How to benchmark methods for structure-based virtual screening of large compound libraries.

Structure-based virtual screening is a useful computational technique for ligand discovery. To systematically evaluate different docking approaches, it is important to have a consistent benchmarking protocol that is both relevant and unbiased. Here, we describe the designing of a benchmarking data set for docking screen assessment, a standard docking screening process, and the analysis and presentation of the enrichment of annotated ligands among a background decoy database.

Theoretical studies of the base pair fidelity of selenium-modified DNA.

The introduction of selenium into DNA in the place of oxygen provides a unique opportunity for studying the fidelity of DNA replication, as well as providing advantages in the growth of DNA crystals and the greater resolution of their structures. However, the atomic mechanisms of the relative stability and base pair recognition of the selenium-modified DNA are poorly understood. In the present study, quantum mechanics calculations were performed on base pairings, base stacking, and base-water interactions for both unmodified thymine and thymine with the 2-exo-oxygen replaced with selenium, and the results were used to develop and validate CHARMM force field parameters for the 2-Se-thymine. Subsequently, molecular dynamics simulations and free-energy perturbation calculations were performed on 11-base DNA sequences containing native thymine and the 2-Se-thymine. The calculated relative free-energy values are in good agreement with experimentally determined relative stability, where the 2-Se-thymine offers similar stability to T-A in cognate DNA, while it dramatically destabilizes the DNA containing the T-G mismatch base pair when 2-Se-thymine is incorporated. Thus, 2-Se-thymine largely increases the native T-A base pair fidelity by discouraging the T-G wobble pair. Insights into the high pairing specificity and the relative stability of selenium-modified DNA were obtained based on detailed structural and energetic analysis of molecular dynamics trajectories. Our studies move one step further toward an understanding of the high base pair fidelity and thermodynamic properties of Se-DNA in solution and in protein-DNA complexes.

Binding-site assessment by virtual fragment screening.

The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules) would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock approximately 11,000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors.

Automated docking screens: a feasibility study.

Molecular docking is the most practical approach to leverage protein structure for ligand discovery, but the technique retains important liabilities that make it challenging to deploy on a large scale. We have therefore created an expert system, DOCK Blaster, to investigate the feasibility of full automation. The method requires a PDB code, sometimes with a ligand structure, and from that alone can launch a full screen of large libraries. A critical feature is self-assessment, which estimates the anticipated reliability of the automated screening results using pose fidelity and enrichment. Against common benchmarks, DOCK Blaster recapitulates the crystal ligand pose within 2 A rmsd 50-60% of the time; inferior to an expert, but respectrable. Half the time the ligand also ranked among the top 5% of 100 physically matched decoys chosen on the fly. Further tests were undertaken culminating in a study of 7755 eligible PDB structures. In 1398 cases, the redocked ligand ranked in the top 5% of 100 property-matched decoys while also posing within 2 A rmsd, suggesting that unsupervised prospective docking is viable. DOCK Blaster is available at http://blaster.docking.org .

Physics-based scoring of protein-ligand complexes: enrichment of known inhibitors in large-scale virtual screening.

We demonstrate that using an all-atom molecular mechanics force field combined with an implicit solvent model for scoring protein-ligand complexes is a promising approach for improving inhibitor enrichment in the virtual screening of large compound databases. The rescoring method is evaluated by the extent to which known binders for nine diverse, therapeutically relevant enzymes are enriched against a background of approximately 100,000 drug-like decoys. The improvement in enrichment is most robust and dramatic within the top 1% of the ranked database, that is, the first thousand compounds; below the first few percent of the ranked database, there is little overall improvement. The improved early enrichment is likely due to the more realistic treatment of ligand and receptor desolvation in the rescoring procedure. We also present anecdotal but encouraging results assessing the ability of the rescoring method to predict specificity of inhibitors for structurally related proteins.

[Molecular design and biological activity of BCR-ABL tyrosine kinase inhibitors].

OBJECTIVE: To discover BCR-ABL tyrosine kinase inhibitors through structure based virtual screening. METHODS: Docking screening against the distinctive inactive conformation of the catalytic domain of BCR-ABL tyrosine kinase was performed on 3D database. The MTT assay was performed to assess the viability of the tumor cells treated with selected compounds. The amount and kinase activity of BCR-ABL protein were detected in the presence of compounds by Western blot analysis and immunoprecipitation. RESULTS: From the top 1,000 compounds with the best DOCK energy score, 15 compounds were selected for biological assay. Eight out of 15 compounds showed notable inhibitory activity against Ph+ human K562 cells with IC50 values ranging from 10 to 200 micromol/L. In cell-based assays of ABL tyrosine phosphorylation, the ability of two kinds of novel, structurally diverse, lead compounds to inhibit ABL kinase activity was observed. However, no significant differences in the amount of BCR-ABL protein were noted on the ABL immunoblot in the presence of these lead compounds. CONCLUSIONS: Two promising lead compounds were discovered to inhibit BCR-ABL tyrosine kinase activity. Virtual screening technique has been proven to narrow down the size of screening compound libraries to the most prospective drug candidates with high success rates.

Identification of novel inhibitors of BCR-ABL tyrosine kinase via virtual screening.

Inhibition of BCR-ABL tyrosine kinase activity has shown to be essential for the treatment of chronic myelogenous leukemia (CML). However, drug resistance has quickly arisen in recent clinical trials for STI571 (Gleevec), which is the first approved drug of CML by inhibiting ABL tyrosine kinase. It is desirable to develop new types of ABL tyrosine kinase inhibitors that may overcome this drug resistance problem. Here we present the discovery of novel inhibitors targeted at the catalytic domain of ABL tyrosine kinase by using three-dimensional database searching techniques. From a database containing 200,000 commercially available compounds, the top 1000 compounds with the best DOCK energy score were selected and subjected to structural diversity and drug likeness analysis, 15 compounds were submitted for biological assay. Eight out of the 15 showed inhibitory activity against K562 cells with IC(50) value ranging from 10 to 200 microM. Two promising compounds showed inhibition in further ABL tyrosine phosphorylation assay. It is anticipated that those two compounds can serve as lead compounds for further drug design and optimization.

Consideration of molecular weight during compound selection in virtual target-based database screening.

Virtual database screening allows for millions of chemical compounds to be computationally selected based on structural complimentary to known inhibitors or to a target binding site on a biological macromolecule. Compound selection in virtual database screening when targeting a biological macromolecule is typically based on the interaction energy between the chemical compound and the target macromolecule. In the present study it is shown that this approach is biased toward the selection of high molecular weight compounds due to the contribution of the compound size to the energy score. To account for molecular weight during energy based screening, we propose normalization strategies based on the total number of heavy atoms in the chemical compounds being screened. This approach is computationally efficient and produces molecular weight distributions of selected compounds that can be selected to be (1) lower than that of the original database used in the virtual screening, which may be desirable for selection of leadlike compounds or (2) similar to that of the original database, which may be desirable for the selection of drug-like compounds. By eliminating the bias in target-based database screening toward higher molecular weight compounds it is anticipated that the proposed procedure will enhance the success rate of computer-aided drug design.