Combinatorial computational approaches to identify tetracycline derivatives as flavivirus inhibitors

Limited structural information of drug targets, cellular toxicity possessed by lead compounds, and large amounts of potential leads are the major issues facing the design-oriented approach of discovering new leads. In an attempt to tackle these issues, we have developed a process of virtual screening based on the observation that conformational rearrangements of the dengue virus envelope protein are essential for the mediation of viral entry into host cells via membrane fusion. Screening was based solely on the structural information of the Dengue virus envelope protein and was focused on a target site that is presumably important for the conformational rearrangements necessary for viral entry. To circumvent the issue of lead compound toxicity, we performed screening based on molecular docking using structural databases of medical compounds. To enhance the identification of hits, we further categorized and selected candidates according to their novel structural characteristics. Finally, the selected candidates were subjected to a biological validation assay to assess inhibition of Dengue virus propagation in mammalian host cells using a plaque formation assay. Among the 10 compounds examined, rolitetracycline and doxycycline significantly inhibited plaque formation, demonstrating their inhibitory effect on dengue virus propagation. Both compounds were tetracycline derivatives with IC(50)s estimated to be 67.1 microM and 55.6 microM, respectively. Their docked conformations displayed common hydrophobic interactions with critical residues that affected membrane fusion during viral entry. These interactions will therefore position the tetracyclic ring moieties of both inhibitors to bind firmly to the target and, subsequently, disrupt conformational rearrangement and block viral entry. This process can be applied to other drug targets in which conformational rearrangement is critical to function.     

Tetralones and flavonoids from Pyrola calliantha

Two new tetralones, pyrolones A (1) and B (2), and a new flavonol glycoside, 2'-O-(4-hydroxybenzoyl)hyperin (3), were isolated from Pyrola calliantha (whole plant), together with six structurally related compounds, including 2'-O-galloylhyperin (4), hyperin (5), formononetin (6), quercetin 3-O-alpha-L-arabinopyranoside (7), quercetin 3-O-alpha-L-arabinofuranoside (8), and kaempferol 3-O-beta-D-galactopyranoside (9). The structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic (UV, ORD, CD, NMR) and mass-spectrometric (HR-ESI-MS) analyses.     

Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.     

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales,Rhodophyta) from East Asia

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.     

Active Transport of Membrane Components by Self-Organization of the Min Proteins

Heterogeneous distribution of components in the biological membrane is critical in the process of cell polarization. However, little is known about the mechanisms that can generate and maintain the heterogeneous distribution of the membrane components. Here, we report that the propagating wave patterns of the bacterial Min proteins can impose steric pressure on the membrane, resulting in transport and directional accumulation of the component in the membrane. Therefore, the membrane component waves represent transport of the component in the membrane that is caused by the steric pressure gradient induced by the differential levels of binding and dissociation of the Min proteins in the propagating waves on the membrane surface. The diffusivity, majorly influenced by the membrane anchor of the component, and the repulsed ability, majorly influenced by the steric property of the membrane component, determine the differential spatial distribution of the membrane component. Thus, transportation of the membrane component by the Min proteins follows a simple physical principle, which resembles a linear peristaltic pumping process, to selectively segregate and maintain heterogeneous distribution of materials in the membrane.

Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.