Benzimidazole- and benzoxazole-based inhibitors of Rho kinase

Inhibitors of Rho kinase have been developed based on two distinct scaffolds, benzimidazoles, and benzoxazoles. SAR studies and efforts to optimize the initial lead compounds are described. Novel selective inhibitors of ROCK-II with excellent potency in both enzyme and cell-based assays were obtained. These inhibitors possess good microsomal stability, low cytochrome P-450 inhibitions and good oral bioavailability.     

Chroman-3-amides as potent Rho kinase inhibitors

Inhibition of Rho kinase (ROCK) is an attractive strategy for the treatment of diseases such as hypertension, glaucoma, and cancer. Here we report chroman-3-amides as highly potent ROCK inhibitors with sufficient kinase selectivity, excellent cell activity, good microsomal stability, and desirable pharmacokinetic properties for study as potential therapeutic agents.     

The PDZ protein Canoe regulates the asymmetric division of Drosophila neuroblasts and muscle progenitors

Asymmetric cell division is a conserved mechanism to generate cellular diversity during animal development and a key process in cancer and stem cell biology. Despite the increasing number of proteins characterized, the complex network of proteins interactions established during asymmetric cell division is still poorly understood. This suggests that additional components must be contributing to orchestrate all the events underlying this tightly modulated process. The PDZ protein Canoe (Cno) and its mammalian counterparts AF-6 and Afadin are critical to regulate intracellular signaling and to organize cell junctions throughout development. Here, we show that Cno functions as a new effector of the apical proteins Inscuteable (Insc)-Partner of Inscuteable (Pins)-Galphai during the asymmetric division of Drosophila neuroblasts (NBs). Cno localizes apically in metaphase NBs and coimmunoprecipitates with Pins in vivo. Furthermore, Cno functionally interacts with the apical proteins Insc, Galphai, and Mushroom body defect (Mud) to generate correct neuronal lineages. Failures in muscle and heart lineages are also detected in cno mutant embryos. Our results strongly support a new function for Cno regulating key processes during asymmetric NB division: the localization of cell-fate determinants, the orientation of the mitotic spindle, and the generation of unequal-sized daughter cells.     

Foldon-guided self-assembly of ultra-stable protein fibers

A common objective in protein engineering is the enhancement of the thermodynamic properties of recombinant proteins for possible applications in nanobiotechnology. The performance of proteins can be improved by the rational design of chimeras that contain structural elements with the desired properties, thus resulting in a more effective exploitation of protein folds designed by nature. In this paper, we report the design and characterization of an ultra-stable self-refolding protein fiber, which rapidly reassembles in solution after denaturation induced by harsh chemical treatment or high temperature. This engineered protein fiber was constructed on the molecular framework of bacteriophage P22 tail needle gp26, by fusing its helical core to the foldon domain of phage T4 fibritin. Using protein engineering, we rationally permuted the foldon upstream and downstream from the gp26 helical core and characterized gp26-foldon chimeras by biophysical analysis. Our data demonstrate that one specific protein chimera containing the foldon immediately downstream from the gp26 helical core, gp26(1-140)-F, displays the highest thermodynamic and structural stability and refolds spontaneously in solution following denaturation. The gp26-foldon chimeric fiber remains stable in 6.0 M guanidine hydrochloride, or at 80 degrees C, rapidly refolds after denaturation, and has both N and C termini accessible for chemical/biological modification, thereby representing an ideal platform for the design of self-assembling nanoblocks.     

Inhibition of oxidative metabolism of tocopherols with omega-N-heterocyclic derivatives of vitamin E

The oxidative metabolism of tocopherols and tocotrienols by monooxygenases is a key factor in the plasma and tissue clearance of forms of vitamin E other than alpha-tocopherol. It is well known that a commonly ingested form of vitamin E, gamma-tocopherol, has greatly reduced plasma half-life (faster clearance) than alpha-tocopherol. The tocotrienols are metabolized even faster than gamma-tocopherol. Both gamma-tocopherol and alpha- and delta-tocotrienol possess intriguing biological activities that are different from alpha-tocopherol, making them potentially of interest for therapeutic use. Unfortunately, the fast clearance of non-alpha-tocopherols from animal tissues is a significant hurdle to maximizing their effect(s) as dietary supplements. We report here the design and synthesis of N-heterocycle-containing analogues of alpha-tocopherol that act as inhibitors of Cyp4F2, the key monooxygenase responsible for omega-hydroxylation of the side chain of tocols. In particular, an omega-imidazole containing compound, 1, [(R)-2-(9-(1H-imidazol-1-yl)nonyl)-2,5,7,8-tetramethylchroman-6-ol] had an ED(50) for inhibition of gamma-CEHC production from gamma-tocopherol of approximately 1 nM when tested in HepG2 cells in culture. Furthermore, feeding of 1 to mice along with rapidly metabolized delta-tocopherol, resulted in a doubling of the delta-tocopherol/alpha-tocopherol ratio in liver (P<0.05). Thus, 1 may be a useful adjuvant to the therapeutic use of non-alpha-tocopherols.     

Response of HEK293 and CHO cells overexpressing fusiogenic syncytin-1 to mitochondrion-mediated apoptosis induced by antimycin A

Apoptosis is essential for the regulation of cellular homeostasis in the placenta and is also involved in the pathophysiology of pregnancy-related diseases such as pre-eclampsia and intrauterine growth restriction (IUGR). Syncytin-1, a fusiogenic glycoprotein of endogenous-retroviral origin expressed in human trophoblasts, facilitates placental syncytium formation and is found reduced in pre-eclamptic placentas. We focus here on the mitochondrial apoptotic pathway and investigate whether the overexpression of syncytin-1 in HEK293-52 (human embryonic kidney cells) and CHO-52 cells influences the apoptotic response to the mitochondrial inhibitor antimycin A (AA). After the induction of apoptosis by 5 microM AA and incubation for up to 36 h in the absence of serum, the mean apoptotic rate was reduced by 15-30% in syncytin-1 transfected cells compared with mock-transfectants. After 12 h of challenge with AA we found lower cytochrome c levels in the cytoplasmic protein fraction and higher amounts in the mitochondrial fraction in syncytin-1 transfectants compared with mock-transfectants. We observed a decreased Mitotracker Red staining of mitochondria following AA challenge for 24 h in mock-treated CHO cells, in particular, compared with syncytin-1 transfectants. Moreover, we found a reduced activation of caspase 9 in syncytin-1 transfected HEK293-52 cells after 48 h of apoptotic challenge compared to mock-transfectants. However, a high expression of anti-apoptotic Bcl-x(L) was found in both cell types. Using syncytin-1 transfected HEK293-52 cells and CHO-52 cells, we provide initial evidence that syncytin-1 may exert its anti-apoptotic function at the mitochondrial level. A reduced release of cytochrome c followed by a diminished activation of caspase 9 is a possible mechanism.     

Abundance,distribution, and territory areas of rock-dwelling Lake Tanganyika cichlid fish species

Lake Tanganyika, the second-oldest and second-deepest lake in the world, harbors an impressive cichlid fish fauna counting about 250 endemic species that are characterized by a great level of ecological, morphological, and behavioral specialization. This study describes and compares cichlid fish communities at two rocky shores with differential human impact in the south of Lake Tanganyika. Species inventories and depth-dependent abundances were elaborated. About 41 and 46 sympatric cichlid species were recorded in the two study sites, respectively. Variabilichromis moorii was the most abundant species (29–60% of total number of fishes), followed by Aulonocranus dewindti (3–19%), Tropheus moorii (12%), Ophthalmotilapia ventralis (4–10%), Eretmodus cyanostictus (6–11%), and Cyathopharynx furcifer (0.01–9%). All other species had abundances below 5%. It further emerged that large cichlids such as Petrochromis species, Cyathopharynx furcifer, and Lobochilotes labiatus were very rare at one location, with frequencies of 0.55% or less. Territorial sizes of three particularly abundant species, Variabilichromis moorii, Aulonocranus dewindti, and Tropheus moorii, were assessed by behavioral observations. We distinguished between territorial core areas and total defended area, yielding average core areas between 0.4 (V. moorii) and 1.6 m² (T. moorii), and total defended areas averaging for each species between 1.6 (V. moorii) and 5.0 m² (A. dewindti) with no significant differences between the two study sites. The data on individual densities are also relevant for evolutionary studies, in that they allow more accurate calculations of effective population sizes.     

Lawsonia intracellularis contains a gene encoding a functional rickettsia-like ATP/ADP translocase for host exploitation

ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [NTT1(Li)]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of NTT1(Li) were determined by measuring the uptake of radioactively labeled substrates by E. coli. NTT1(Li) transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 (+/- 36.5) microM for ATP and 275.2 (+/- 28.1) microM for ADP, identifying this protein as a functional ATP/ADP translocase. NTT1(Li) is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors.     

Clp-dependent proteolysis down-regulates central metabolic pathways in glucose-starved Bacillus subtilis

Entry into stationary phase in Bacillus subtilis is linked not only to a redirection of the gene expression program but also to posttranslational events such as protein degradation. Using ³⁵S-labeled methionine pulse-chase labeling and two-dimensional polyacrylamide gel electrophoresis we monitored the intracellular proteolysis pattern during glucose starvation. Approximately 200 protein spots diminished in the wild-type cells during an 8-h time course. The degradation rate of at least 80 proteins was significantly reduced in clpP, clpC, and clpX mutant strains. Enzymes of amino acid and nucleotide metabolism were overrepresented among these Clp substrate candidates. Notably, several first-committed-step enzymes for biosynthesis of aromatic and branched-chain amino acids, cell wall precursors, purines, and pyrimidines appeared as putative Clp substrates. Radioimmunoprecipitation demonstrated GlmS, IlvB, PurF, and PyrB to be novel ClpCP targets. Our data imply that Clp proteases down-regulate central metabolic pathways upon entry into a nongrowing state and thus contribute to the adaptation to nutrient starvation. Proteins that are obviously nonfunctional, unprotected, or even “unemployed” seem to be recognized and proteolyzed by Clp proteases when the resources for growth become limited.