Atomically precise organomimetic cluster nanomolecules assembled via perfluoroaryl-thiol SNAr chemistry.

The majority of biomolecules are intrinsically atomically precise, an important characteristic that enables rational engineering of their recognition and binding properties. However, imparting a similar precision to hybrid nanoparticles has been challenging because of the inherent limitations of existing chemical methods and building blocks. Here we report a new approach to form atomically precise and highly tunable hybrid nanomolecules with well-defined three-dimensionality. Perfunctionalization of atomically precise clusters with pentafluoroaryl-terminated linkers produces size-tunable rigid cluster nanomolecules. These species are amenable to facile modification with a variety of thiol-containing molecules and macromolecules. Assembly proceeds at room temperature within hours under mild conditions, and the resulting nanomolecules exhibit high stabilities because of their full covalency. We further demonstrate how these nanomolecules grafted with saccharides can exhibit dramatically improved binding affinity towards a protein. Ultimately, the developed strategy allows the rapid generation of precise molecular assemblies to investigate multivalent interactions.

Oxidative in vitro metabolism of liquiritigenin, a bioactive compound isolated from the Chinese herbal selective estrogen beta-receptor agonist MF101.

Liquiritigenin [2,3-dihydro-7-hydroxy-2-(4-hydroxyphenyl)-(S)-4H-1-benzopyran-4-one] is one of the major active compounds of MF101, an herbal extract currently in clinical trials for the treatment of hot flashes and night sweats in postmenopausal women. MF101 is a selective estrogen receptor beta agonist but does not activate the estrogen receptor alpha. Incubation with pooled human liver microsomes yielded a single metabolite. Its structure was elucidated using tandem mass spectrometry in combination with analysis of the fragmentation patterns. The metabolite resulted from the loss of two hydrogens and rearrangement to the stable 7,4'-dihydroxyflavone. The structure was also confirmed by comparison with authentic standard material. Maximum apparent reaction velocity (V(max)) and Michaelis-Menten constant (K(m)) for the formation of 7,4'-dihydroxyflavone were 32.5 nmol/g protein/min and 128 microM, respectively. After correction for protein binding (free fraction = 0.84), the apparent intrinsic clearance (CL(int)) for 7,4'-dihydroxyflavone formation was 0.3 ml/g/min. Liquiritigenin was almost exclusively metabolized by CYP3A enzymes. Comparison of liquiritigenin metabolism in human liver microsomes isolated from 16 individuals showed 9.5-fold variability in metabolite formation (3.4-32.2 nmol/g protein/min). An estrogen receptor luciferase assay indicated that the metabolite was a 3-fold more potent activator of the estrogen receptor beta than the parent compound and did not activate the estrogen receptor alpha.

Inhibition of Cdc7/Dbf4 kinase activity affects specific phosphorylation sites on MCM2 in cancer cells.

The Cdc7/Dbf4 kinase is required for initiation of DNA replication and also plays a role in checkpoint function in response to replication stress. Exactly how Cdc7/Dbf4 mediates those activities remains to be elucidated. Cdc7/Dbf4 physically interacts with and phosphorylates the minichromosome maintenance complex (MCM), such as MCM2, MCM4 and MCM6. Cdc7/Dbf4 activity is required for association of Cdc45 followed by recruitment of DNA polymerase on the chromatin. Using high resolution mass spectrometry, we identified six phosphorylation sites on MCM2, two of them have not been described before. We provide evidence that Cdc7/Dbf4 mediates phosphorylation on serine 108 and serine 40 on human MCM2 in vitro and in vivo in cancer cells in the absence of DNA damage. Antibodies specific to pS108 or pS40 confirmed the sites and established useful read-outs for inhibition of Cdc7/Dbf4. This report demonstrates the utility of an in vitro to in vivo workflow utilizing immunoprecipitation and mass spectrometry to map phosphorylation sites on endogenous kinase substrates. The approach can be readily generalized to identify target modulation read-outs for other potential kinase cancer targets.

Liquiritigenin is a plant-derived highly selective estrogen receptor beta agonist.

After the Women's Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERbeta-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERbeta-selective properties. In this study we isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERbeta but not ERalpha. The ERbeta-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERbeta.

State-dependent GABAergic inhibition of sciatic nerve-evoked responses of dorsal spinocerebellar tract neurons.

Peripheral nerve-evoked potentials recorded in the cerebellum 35 yr ago inferred that sensory transmission via the dorsal spinocerebellar tract (DSCT) is reduced occasionally and only during eye movements of active sleep compared with wakefulness or quiet sleep. A reduction or withdrawal of primary afferent input and/or ongoing inhibition of individual lumbar DSCT neurons may underlie this occurrence. This study distinguished between these possibilities by examining whether peripheral nerve-evoked responses recorded from individual DSCT neurons are suppressed specifically during active sleep, and if so, whether GABA mediates this phenomenon. Synaptic responses to threshold stimuli applied to the sciatic nerve were characterized by a single spike response at short latency and/or a longer latency burst of action potentials. During the state of quiet wakefulness, response magnitude did not differ from that observed during quiet sleep. During active sleep, short and long latency responses were suppressed by 26 and 14%, respectively, and returned to pre-active sleep levels following awakening from active sleep. Sciatic nerve-evoked early and late responses were further analyzed in a paired fashion around computer-tagged eye movement events that hallmark the state of active sleep. Response magnitude was suppressed by 14.4 and 11.5%, respectively, during eye movement events of active sleep. The GABA(A) antagonist bicuculline, applied juxtacellularly by microiontophoresis, abolished response suppression during non-eye movement periods and eye movement events of active sleep. In conclusion, synaptic transmission via DSCT neurons is inhibited by GABA tonically during non-eye movement periods and phasically during eye movement events of active sleep.

Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells.

It has been shown that rat aortic smooth muscle cells (AoSMCs) lost PKG-I expression when propagated repetitively or grown at low densities. Conversely, AoSMCs isolated from PKG-I deficient mice are indistinguishable from those isolated from normal mice in morphology and growth characteristics. In this study, human AoSMCs were grown from passage 9 (p9) to passage 15 (p15) and rat AoSMCs were isolated and cultured from p1 through p15. Western blotting and immunofluorescence microscopy showed little difference in PKG-I expression among different passages. Next, rat AoSMCs of p4 were grown and harvested at different cell densities. Western blotting again showed little difference among cells seeded or harvested at different densities. To test the effect of cell passage on PKG-I activation, rat AoSMCs of p4 and p11 were treated with cGMP and analyzed by Western blotting for phosphorylated vasodilator-stimulated phosphoprotein (P-VASP). The results showed that p4 had higher level of PKG-I activation than p11.