Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits.

The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine.

Substituted pyrazoles as novel sEH antagonist: investigation of key binding interactions within the catalytic domain.

A novel series of pyrazole sEH inhibitors is reported. Lead optimization efforts to replace the aniline core are also described. In particular, 2-pyridine, 3-pyridine and pyridazine analogs are potent sEH inhibitors with favorable CYP3A4 inhibitory and microsomal stability profiles.

Compensatory mechanism for homeostatic blood pressure regulation in Ephx2 gene-disrupted mice.

Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase- and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene.

Assembly of major histocompatibility complex (MHC) class II transcription factors: association and promoter recognition of RFX proteins.

Major histocompatibility complex (MHC) class II genes are regulated at the transcriptional level by coordinate action of a limited number of transcription factors that include regulatory factor X (RFX), class II transcriptional activator (CIITA), nuclear factor Y (NF-Y), and cyclic AMP-response element binding protein (CREB). Here, the MHC class-II-specific transcription factors and CREB were expressed in insect cells with recombinant baculoviruses, isolated, and characterized by biochemical and biophysical methods. Analytical ultracentrifugation (AUC) has demonstrated that RFX is a heterotrimer. A heterodimer of RFX5 and RFX-AP was also observed. A high-affinity interaction (K(d) = 25 nM) between RFX5 and RFX-AP was measured by isothermal titration calorimetry (ITC), while the interaction between RFX-AP and RFX-ANK is at least an order of magnitude weaker. The biophysical data show that the interaction between RFX-AP and RFX5 is a key event in the assembly of the heterotrimer. Fluorescence anisotropy was used to determine protein-nucleic acid binding affinities for the RFX subunits and complexes binding to duplex DNA. The RFX5 subunit was found to drive recognition of the promoter, while the auxiliary RFX-AP and RFX-ANK subunits were shown to contribute to the specificity of binding for the overall complex. AUC experiments demonstrate that in the absence of additional subunits, monomeric RFX5 binds to X-box DNA with a 1:1 stoichiometry. Interactions between CREB, CIITA, and RFX in the absence of DNA were demonstrated using bead-based immunoprecipitation assays, confirming that preassociation with DNA is not required for forming the macromolecular assemblies that drive MHC class II gene expression.

Catalysis and function of the p38 alpha.MK2a signaling complex.

The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.

Thermal denaturation: a method to rank slow binding, high-affinity P38alpha MAP kinase inhibitors.

It has been reported that the diaryl urea class of p38alpha inhibitors binds to p38 map kinase with both high affinity and slow binding kinetics (Pargellis et al. Nat. Struct. Biol. 2002, 9, 268-272). The slow binding kinetics of this class of inhibitors is believed to be the result of binding to an allosteric pocket adjacent to the p38alpha active site. The use of traditional kinetic and equilibrium methods to measure the binding affinity of this class of compounds has created many challenges for determination of structure-activity relationships (SAR). The thermal denaturation method provides a means of measuring high-affinity interactions. In this paper, the method of thermal denaturation will be described as it has been applied to the diaryl urea class of p38 map kinase inhibitors.

14,15-Dihydroxyeicosatrienoic acid relaxes bovine coronary arteries by activation of K(Ca) channels.

Epoxyeicosatrienoic acids (EETs) cause vascular relaxation by activating smooth muscle large conductance Ca(2+)-activated K(+) (K(Ca)) channels. EETs are metabolized to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase. We examined the contribution of 14,15-DHET to 14,15-EET-induced relaxations and characterized its mechanism of action. 14,15-DHET relaxed U-46619-precontracted bovine coronary artery rings but was approximately fivefold less potent than 14,15-EET. The relaxations were inhibited by charybdotoxin, iberiotoxin, and increasing extracellular K(+) to 20 mM. In isolated smooth muscle cells, 14,15-DHET increased an iberiotoxin-sensitive, outward K(+) current and increased K(Ca) channel activity in cell-attached patches and inside-out patches only when GTP was present. 14,15-[(14)C]EET methyl ester (Me) was converted to 14,15-[(14)C]DHET-Me, 14,15-[(14)C]DHET, and 14,15-[(14)C]EET by coronary arterial rings and endothelial cells but not by smooth muscle cells. The metabolism to 14,15-DHET was inhibited by the epoxide hydrolase inhibitors 4-phenylchalcone oxide (4-PCO) and BIRD-0826. Neither inhibitor altered relaxations to acetylcholine, whereas relaxations to 14,15-EET-Me were increased slightly by BIRD-0826 but not by 4-PCO. 14,15-DHET relaxes coronary arteries through activation of K(Ca) channels. Endothelial cells, but not smooth muscle cells, convert EETs to DHETs, and this conversion results in a loss of vasodilator activity.