Probing chelation motifs in HIV integrase inhibitors

A series of HIV integrase (HIV-1 IN) inhibitors were synthesized to evaluate the role of the metal-binding group (MBG) in this class of metalloenzyme inhibitors. A total of 21 different raltegravir-chelator derivative (RCD) compounds were prepared that differed only in the nature of the MBG. These IN strand-transfer inhibitors (INSTIs) were evaluated in vitro in cell-free enzyme activity assays, and the in vitro results were further validated in cell culture experiments. All of the active compounds showed selective inhibition of the strand-transfer reaction over 3'-processing, suggesting a common mode of action with raltegravir. The results of the in vitro activity suggest that the nature of the MBG donor atoms, the overall MBG structure, and the specific arrangement of the MBG donor atom triad are essential for obtaining maximal HIV-1 IN inhibition. At least two compounds (RCD-4, RCD-5) containing a hydroxypyrone MBG were found to display superior strand-transfer inhibition when compared to an abbreviated analogue of raltegravir (RCD-1). By isolating and examining the role of the MBG in a series of INSTIs, we have identified a scaffold (hydroxypyrones) that may provide access to a unique class of HIV-1 IN inhibitors, and may help overcome rising raltegravir resistance.     

Human fibrocytes coexpress thyroglobulin and thyrotropin receptor

Thyroglobulin (Tg) is the macromolecular precursor of thyroid hormones and is thought to be uniquely expressed by thyroid epithelial cells. Tg and the thyroid-stimulating hormone receptor (TSHR) are targets for autoantibody generation in the autoimmune disorder Graves disease (GD). Fully expressed GD is characterized by thyroid overactivity and orbital tissue inflammation and remodeling. This process is known as thyroid-associated ophthalmopathy (TAO). Early reports suggested that in TAO, both Tg and TSHR become overexpressed in orbital tissues. Previously, we found that CD34(+) progenitor cells, known as fibrocytes, express functional TSHR, infiltrate the orbit, and comprise a large subset of orbital fibroblasts in TAO. We now report that fibrocytes also express Tg, which resolves as a 305-kDa protein on Western blots. It can be immunoprecipitated with anti-Tg Abs. Further, (125)iodine and [(35)S]methionine are incorporated into Tg expressed by fibrocytes. De novo Tg synthesis is attenuated with a specific small interfering RNA targeting the protein. A fragment of the Tg gene promoter fused to a luciferase reporter exhibits substantial activity when transfected into fibrocytes. Unlike fibrocytes, GD orbital fibroblasts, which comprise a mixture of CD34(+) and CD34(-) cells, express much lower levels of Tg and TSHR. When sorted into pure CD34(+) and CD34(-) subsets, Tg and TSHR mRNA levels become substantially higher in CD34(+) cells. These findings indicate that human fibrocytes express multiple "thyroid-specific" proteins, the levels of which are reduced after they infiltrate tissue. Our observations establish the basis for Tg accumulation in orbital GD.     

Aberrant S-nitrosylation mediates calcium-triggered ventricular arrhythmia in the intact heart

Nitric oxide (NO) derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca²⁺ handling proteins. Deficient S-nitrosylation of the cardiac ryanodine receptor (RyR2) has a variable effect on SR Ca²⁺ leak/sparks in isolated myocytes, likely dependent on the underlying physiological state. It remains unknown, however, whether such molecular aberrancies are causally related to arrhythmogenesis in the intact heart. Here we show in the intact heart, reduced NOS1 activity increased Ca²⁺-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca²⁺]_i. These arrhythmias arose from increased spontaneous SR Ca²⁺ release, resulting from a combination of decreased RyR2 S-nitrosylation (RyR2-SNO) and increased RyR2 oxidation (RyR-SOx) (i.e., increased reactive oxygen species (ROS) from xanthine oxidoreductase activity) and could be suppressed with xanthine oxidoreductase (XOR) inhibition (i.e., allopurinol) or nitric oxide donors (i.e., S-nitrosoglutathione, GSNO). Surprisingly, we found evidence of NOS1 down-regulation of RyR2 phosphorylation at the Ca²⁺/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between nitrosylation and phosphorylation of RyR2. Finally, we show that nitroso–redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype by oxidative stress. Our findings suggest that nitroso–redox imbalance is an important mechanism of ventricular arrhythmias in the intact heart under disease conditions (i.e., elevated [Ca²⁺]_i and oxidative stress), and that therapies restoring nitroso–redox balance in the heart could prevent sudden arrhythmic death.Nitric oxide (NO) is an important regulator of cardiac function via both the activation of cyclic guanosine monophosphate-dependent signaling pathways and direct posttranslational modification of protein thiols (S-nitrosylation) (1). NO derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca²⁺ handling proteins (2). In particular, nitrosylation of both skeletal and cardiac muscle ryanodine receptors (RyR1 and RyR2, respectively) alters their release properties, favoring activation (3, 4). Notably, an increase in RyR2 open probability can cause spontaneous SR Ca²⁺ release, which may cause arrhythmias. Recently, it was shown that decreased RyR2 S-nitrosylation (RyR2-SNO) through loss of NOS1, was associated with increased spontaneous SR Ca²⁺ release events in isolated cardiomyocytes, following rapid pacing (5). In a separate study, NOS1 deficiency was shown to decrease spontaneous SR Ca²⁺ sparks and the open probability of RyR2 under resting conditions in cardiomyocytes and lipid bilayers, respectively (6). These studies suggest that NOS1 deficiency has a variable effect on RyR2 function, likely dependent on the underlying physiological state (i.e., rapid heart rate versus quiescence). It remains unknown, however, whether these changes create a substrate for arrhythmogenesis in the intact heart.It is increasingly evident that activities of nitric oxide and reactive oxygen species (ROS) are tightly coupled in cardiomyocytes producing nitroso–redox balance. Elevated ROS production (oxidative stress) is a hallmark of several cardiovascular diseases associated with increased risk of fatal ventricular arrhythmias [e.g., myocardial infarction (MI) and heart failure]. Burger et al. (7) recently demonstrated an increased incidence of ventricular arrhythmias following MI in NOS1-deficient mice. These data suggest that a nitroso–redox imbalance may be arrhythmogenic in the setting of MI. However, the molecular basis of the increased arrhythmogenesis is not known.In the current study, we found that decreased NOS1 activity increased Ca²⁺-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca²⁺]_i. These arrhythmias arose from increased spontaneous SR Ca²⁺ release resulting from a combination of decreased RyR2-SNO and increased RyR2 oxidation (RyR2-SOx) [i.e., increased ROS from xanthine oxidoreductase (XOR) activity] and could be suppressed with xanthine oxidoreductase inhibition (i.e., allopurinol) or nitric oxide donors (i.e., GSNO). Notably, we found evidence of NOS1 regulation of RyR2 phosphorylation at the Ca²⁺/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between the nitrosylation and phosphorylation states of RyR2. Finally, we show that nitroso–redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype under oxidative stress.     

Age-related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline

The cognitive decline associated with normal aging was long believed to be due primarily to decreased synaptic density and neuron loss. Recent studies in both humans and non-human primates have challenged this idea, pointing instead to disturbances in white matter (WM) including myelin damage. Here, we review both cross-sectional and longitudinal studies in humans and non-human primates that collectively support the hypothesis that WM disturbances increase with age starting at middle age in humans, that these disturbances contribute to age-related cognitive decline, and that age-related WM changes may occur as a result of free radical damage, degenerative changes in cells in the oligodendrocyte lineage, and changes in microenvironments within WM.