Synthesis and biological evaluation of bicyclic and tricyclic substituted nortropane derivatives: discovery of a novel selective alpha1D-adrenergic receptor ligand

A range of 3,6,6-trisubstituted nortropane derivatives based upon 6beta-phenyltropane-3beta,6 alpha-diol have been synthesised from 6beta-hydroxytropinone, including some novel related tricyclic hemi-ketal and tricyclic ketal compounds. Derivatives were assessed for pharmacological affinity and selectivity at alpha(1)-adrenergic receptors, and 6beta-phenyl-8-azabicyclo[3.2.1]octan-3-spiro-2'-(1',3'-dioxolane)-6-ol, a novel lead compound selective for the alpha(1D)-adrenergic receptor, is reported.     

Tropane alkaloids from the leaves and stem bark of Erythroxylon alaternifolium and Erythroxylon rotundifolium

A novel 3alpha,6beta,7beta-triol tropane alkaloid esterified by two benzoyl residues was isolated from the leaves of the endemic cuban species, Erythroxylon alaternifolium. Another novel 3alpha,6alpha,7beta-triol tropane alkaloid esterified by trimethoxycinnamoyl and trimethoxybenzoyl residues was isolated from the leaves and stem bark of a second endemic cuban species, Erythroxylon rotundifolium. Their structures were elucidated as 3alpha,7beta-dibenzoyloxy-6beta-hydroxy-tropane and 3alpha-(3,4,5-trimethoxycinnamoyloxy)-7beta-(3,4,5- trimethoxybenzoyloxy)-6alpha-hydroxy-tropane by spectroscopic methods including 2D-NMR techniques.     

ARF6 GTPase protects the post-mitotic midbody from 14-3-3-mediated disintegration

In cytokinesis, there is a lengthy interval between cleavage furrow ingression and abscission, during which the midbody microtubule bundle provides both structural support for a narrow intercellular bridge and a platform that orchestrates the biochemical preparations for abscission. It is currently unclear how the midbody structure is stably maintained during this period. Here, we report a novel role for the ADP-ribosylation factor 6 (ARF6) GTPase in the post-mitotic stabilisation of midbody. Centralspindlin kinesin-6/RhoGAP complex, a midbody component critical for both the formation and function of the midbody, assembles in a sharp band at the centre of the structure in a manner antagonised by 14-3-3 protein. We show that ARF6 competes with 14-3-3 for binding to centralspindlin such that midbodies formed by centralspindlin mutants that can bind 14-3-3 but not ARF6 frequently collapse before abscission. These data indicate a novel mechanism for the regulation of midbody dynamics in which ARF6 protects the compacted centralspindlin assembly from dissipation by 14-3-3.     

Synthesis and antiangiogenic activity of 6-amido-2,4,5-trimethylpyridin-3-ols

We recently reported that 6-aminoalkyl-2,4,5-trimethylpyridin-3-ols, novel series of 6-aminopyridin-3-ol-based antioxidants, have high antiangiogenic activities. In pursuit of wider variety in the analogues, we here report the synthesis and antiangiogenic activities of 6-amidoalkyl-2,4,5-trimethylpyridin-3-ols, which would not be considered excellent antioxidants because of the poorer electron-donating effect of the C(6)-amido group than the corresponding C(6)-amino group. The selected 6-amido compounds showed up to several fold-higher antiangiogenic activities and up to an order of magnitude better antitumor activities in the chick embryo chorioallantoic membrane (CAM) assay than SU4312, a positive control. We also found that paracetamol, as a direct phenolic analogue of our simplest 6-amidopyridin-3-ol, showed a moderate level of antiangiogenic activity. We propose this study will offer a basis for a scaffold of novel angiogenesis inhibitors that can perturb angiogenesis-related pathologies.     

Differential and restricted expression of novel collagen VI chains in mouse

Recently, three novel collagen VI chains, α4, α5 and α6, were identified. These are thought to substitute for the collagen VI α3 chain, probably forming α1α2α4, α1α2α5 or α1α2α6 heterotrimers. The expression pattern of the novel chains is so far largely unknown. In the present study, we compared the tissue distribution of the novel collagen VI chains in mouse with that of the α3 chain by immunohistochemistry, immunoelectron microscopy and immunoblots. In contrast to the widely expressed α3 chain, the novel chains show a highly differential, restricted and often complementary expression. The α4 chain is strongly expressed in the intestinal smooth muscle, surrounding the follicles in ovary, and in testis. The α5 chain is present in perimysium and at the neuromuscular junctions in skeletal muscle, in skin, in the kidney glomerulus, in the interfollicular stroma in ovary and in the tunica albuginea of testis. The α6 chain is most abundant in the endomysium and perimysium of skeletal muscle and in myocard. Immunoelectron microscopy of skeletal muscle localized the α6 chain to the reticular lamina of muscle fibers. The highly differential and restricted expression points to the possibility of tissue-specific roles of the novel chains in collagen VI assembly and function.     

Isolation of five new 5 alpha-hydroxy-6-keto-delta 7 sterols from the marine sponge Oscarella lobularis

Five novel sterols isolated from the marine sponge Oscarella lobularis have been identified on the basis of spectral arguments: cholest-7-ene-3beta,5alpha-diol-6-one (1), cholesta-7,22E-diene-3beta, 5alpha-diol-6-one (2), 24-methylcholesta-7,22E-diene-3beta,5alpha-diol-6-one (3), 24-methylcholesta-7,24(28)-diene-3beta,5alpha-diol-6-one (4), and 24-ethylcholest-7-ene-3beta,5alpha-diol-6-one (5).     

Glabrizoflavone--a novel isoflavone from Glycyrrhiza glabra L.]

7-O-Methylglabranin, 6-C-prenylpinocembrin, glabranin, pinocembrin, galangin, and a novel isoflavonoid, (E)-5,7,4'-trihydroxy-6-(3-hydroxymethyl-2-butenyl)isoflavone (glabrisoflavone) were isolated from the aerial parts of Glycyrrhiza glabra L. The structure of the novel isoflavonoid was elucidated on the basis of chemical transformations and spectral data.     

Characterization of novel partial t-haplotypes tM1, tM2, tM3, tM4]

The results of genetic analysis of the effects of four novel partial mouse TM-haplotypes are presented in this work. Fertility and viability of tM/tM homozygotes and tM/t6 compounds, transmission ratio distortion (trd) in males heterozygous for tM, suppression of recombination and taillessness effects were studied. Three novel t-haplotypes tM1,2,4 are viable and heterozygous for these haplotypes males T/tM show low trd (20-30%). Comparison of these data and the t6-haplotype structure suggests that the tM1,2,4-haplotypes were derived as a result of recombination events in the non-inverted T-complex fragment located between two inversions. The tM3-haplotype is semilethal and heterozygous T/tM3 males show the trd equal to that of t6-heterozygotes. Homozygous tM3/tM3 and tM3/t6 male compounds are fertile or subfertile. Potential recombination ways of derivation of tM3 are discussed.     

Design,synthesis and evaluation of 1,2-benzisothiazol-3-one derivatives as potent caspase-3 inhibitors

A number of 1,2-benzisothiazol-3-one derivatives were prepared through structural modification of the original compound from high-throughput screening. Some analogues (e.g., 6b, 6r, 6s and 6w) were identified as novel and potent caspase inhibitors with IC₅₀ of nanomolar. Structure–activity relationship (SAR) studies for caspase-3 inhibition were evaluated in vitro. Molecular modeling studies provided further insight into the interaction of this class of compounds with activated caspase-3. The present small molecule caspase-3 inhibitor with novel structures different from structures of known caspase inhibitors revealed a new direction for therapeutic strategies directed against diseases involving abnormally up-regulated apoptosis.     

A novel 6-butyl-3-hydroxyflavanone from heartwood of Bauhinia purpurea

Three glycerol derivatives and a novel 6-butyl-3-hydroxyflavanone derivative were isolated from the heartwood of Bauhinia purpurea L. The latter compound was elucidated as 6-(3″-oxobutyl)taxifolin on the basis of spectral evidence.     

Skeletal myoblasts utilize a novel beta 1-series integrin and not alpha 6 beta 1 for binding to the E8 and T8 fragments of laminin.

The E8 fragment of laminin stimulates myoblast attachment and locomotion. Myoblast attachment to laminin/E8 was blocked by anti-integrin antibodies against beta 1-chains but not by antibodies against alpha 6-chains. By contrast, other cell lines (e.g. B16, HT1080, P19, F9, Pys2, 3T3, and 3T6) were blocked both by anti-beta 1 and anti-alpha 6. All cells tested also bound to approximately 125-kDa C-terminal fragments of E8 (T8 and T8'). Immunoprecipitation of surface-iodinated myoblasts revealed beta 1-, alpha 3-, and alpha 5-integrin chains and a novel chain that co-precipitated with anti-beta 1 antibodies running at approximately 95 kDa (reduced). I125-alpha 6 beta 1 was immunoprecipitated from cells whose attachment to E8 was blocked by anti-alpha 6 antibodies. By contrast, little alpha 6 beta 1 could be immunoprecipitated from myoblasts. beta 1-Integrin and the novel alpha-chain (alpha'), Mr approximately 120,000/approximately 95.000 (nonreduced/reduced), from myoblast lysates were retained during affinity chromatography on Engelbreth-Holm-Swarm-laminin affinity columns. beta 1, alpha 1, and the novel alpha' were retained from Rugli cell lysates on Engelbreth-Holm-Swarm-laminin columns. alpha 3 was not bound. When E8 was used as affinity matrix, only beta 1 and alpha' were retained. The N-terminal sequence of Rugli alpha' was homologous to alpha-chains of beta 1-series integrins and was most similar to alpha 6 (9 identical residues out of 14). However, there were distinctive differences; in particular, 2 residues were deleted in comparison with alpha 6.     

Effect of Behavioural Feedback on Circadian Clocks of the Nocturnal Field Mouse Mus booduga

The effect of 'novel running wheels' on circadian clocks of the nocturnal field mouse Mus booduga was investigated during free-running and entrained conditions. In order to find out whether daily access to novel running wheels can entrain the locomotor activity rhythms experimental animals (n = 6) were provided with 'novel running wheels' at a fixed time of the day. The control animals (n = 5) were handled similar to the experimental animals but were not given access to novel running wheels. The results show that daily access to novel running wheels entrained the free-running locomotor activity rhythm of these mice. The post-entrainment free-running period (τ) of the experimental animals was significantly shorter than the pre-entrainment τ, whereas the pre- and post-treatment τ of the control animals did not differ significantly. In separate set of experiments, the effect of access to novel running wheels on the rate of re-entrainment was studied after a 6 h phase advance/delay in 24 h (12:12 h) light/dark (LD) cycles. Experimental animals were given access to novel running wheels for 3-h, 1 h after the 'lights-off' only on the first day of the 'new LD cycles'. Experimental animals took fewer cycles to re-entrain to 6-h phase advanced LD cycles compared to the control animals. After a phase delay in the LD cycles by 6h, the experimental animals took more number of cycles to re-entrain compared to the control animals. These results thus suggest that access to novel running wheel can act as a Zeitgeber for the circadian clocks of the nocturnal mouse M. booduga, and can also modify the rates of re-entrainment to phase shifted LD cycles, in a time-dependent manner.     

Enhanced Immune Response and Protective Effects of Nano-chitosan-based DNA Vaccine Encoding T Cell Epitopes of Esat-6 and FL against Mycobacterium Tuberculosis Infection

Development of a novel and effective vaccine against Mycobacterium tuberculosis (M.tb) is a challenging for preventing TB infection. In this study, a novel nanoparticle-based recombinant DNA vaccine was developed, which contains Esat-6 three T cell epitopes (Esat-6/3e) and fms-like tyrosine kinase 3 ligand (FL) genes (termed Esat-6/3e-FL), and was enveloped with chitosan (CS) nanoparticles (nano-chitosan). The immunologic and protective efficacy of the nano-chitosan-based DNA vaccine (termed nano-Esat-6/3e-FL) was assessed in C57BL/6 mice after intramuscular prime vaccination with the plasmids DNA and nasal boost with the Esat-6/3e peptides. The results showed that the immunized mice remarkably elicited enhanced T cell responses and protection against M.tb H37Rv challenge. These findings indicate that the nano-chitosan can significantly elevate the immunologic and protective effects of the DNA vaccine, and the nano-Esat-6/3e-FL is a useful vaccine for preventing M.tb infection in mice.     

Selective CDK6 degradation mediated by cereblon,VHL, and novel IAP-recruiting PROTACs

Inhibitors of CDK4 and CDK6 have emerged as important FDA-approved treatment options for breast cancer patients. The properties and pharmacology of CDK4/6 inhibitor medicines have been extensively profiled, and investigations into the degradation of these targets via a PROTAC strategy have also been reported. PROTACs are a novel class of small-molecules that offer the potential for differentiated pharmacology compared to traditional inhibitors by redirecting the cellular ubiquitin–proteasome system to degrade target proteins of interest. We report here the preparation of palbociclib-based PROTACs that incorporate binders for three different E3 ligases, including a novel IAP-binder, which effectively degrade CDK4 and CDK6 in cells. In addition, we show that the palbociclib-based PROTACs in this study that recruit different E3 ligases all exhibit preferential CDK6 vs. CDK4 degradation selectivity despite employing a selection of linkers between the target binder and the E3 ligase binder.     

A Novel TRPC6 Mutation That Causes Childhood FSGS

Background

TRPC6, encoding a member of the transient receptor potential (TRP) superfamily of ion channels, is a calcium-permeable cation channel, which mediates capacitive calcium entry into the cell. Until today, seven different mutations in TRPC6 have been identified as a cause of autosomal-dominant focal segmental glomerulosclerosis (FSGS) in adults.

Methodology/Principal Findings

Here we report a novel TRPC6 mutation that leads to early onset FSGS. We identified one family in whom disease segregated with a novel TRPC6 mutation (M132T), that also affected pediatric individuals as early as nine years of age. Twenty-one pedigrees compatible with an autosomal-dominant mode of inheritance and biopsy-proven FSGS were selected from a worldwide cohort of 550 families with steroid resistant nephrotic syndrome (SRNS). Whole cell current recordings of the mutant TRPC6 channel, compared to the wild-type channel, showed a 3 to 5-fold increase in the average out- and inward TRPC6 current amplitude. The mean inward calcium current of M132T was 10-fold larger than that of wild-type TRPC6. Interestingly, M132T mutants also lacked time-dependent inactivation. Generation of a novel double mutant M132T/N143S did not further augment TRPC6 channel activity.

Conclusions

In summary, our data shows that TRPC6 mediated FSGS can also be found in children. The large increase in channel currents and impaired channel inactivation caused by the M132T mutant leads to an aggressive phenotype that underlines the importance of calcium dose channeled through TRPC6.     

Sequence and structural difference favors a distinct preference of Wnt3a binding with co-receptor LRP6

Wnt signaling pathway plays a key role in a wide array of development and physiological processes. Wnt proteins interact with two different co-receptors LRP5/6 and ROR 2, leading to different signal transductions in the cell. Though the Wnt family of proteins has high sequence similarity the specificity for particular co-receptor is not well understood. The choice of pathway is attributed to the binding of Wnt complex to the co-receptor. Our current study is a novel approach using homology modeling, docking, and structural alignment to unravel the structural differences between Wnt3a and Wnt5b binding to LRP6. The conservation of a protruding loop has been identified in Wnt3a protein indicating an enhanced ability of Wnt3a to bind to LRP5/6 against its counter parts. The docking studies have further substantiated the findings. This could potentially help us design and develop novel inhibitors targeting Wnt3a-LRP6 complex in specific tissues or disease states.     

Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae

The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 --> I(1,3,4,5)P4 --> I(1,2,3,4,5)P5 --> PP-IP4 --> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.