Identification and structure-based optimization of novel dihydropyrones as potent HCV RNA polymerase inhibitors

A novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal structure revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor binding. Herein we report the structure-activity relationships (SARs) of this novel class of dihydropyrone-containing compounds that show potent inhibitory activities against the HCV RNA polymerase in biochemical assays.     

Oligomerization Is Crucial for the Stability and Function of Heme Oxygenase-1 in the Endoplasmic Reticulum

Heme oxygenase-1 (HO-1), a stress-inducible enzyme anchored in the endoplasmic reticulum (ER) by a single transmembrane segment (TMS) located at the C terminus, interacts with NADPH cytochrome P450 reductase and biliverdin reductase to catalyze heme degradation to biliverdin and its metabolite, bilirubin. Previous studies suggested that HO-1 functions as a monomer. Using chemical cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments, here we showed that HO-1 forms dimers/oligomers in the ER. However, oligomerization was not observed with a truncated HO-1 lacking the C-terminal TMS (amino acids 266–285), which exhibited cytosolic and nuclear localization, indicating that the TMS is essential for the self-assembly of HO-1 in the ER. To identify the interface involved in the TMS-TMS interaction, residue Trp-270, predicted by molecular modeling as a potential interfacial residue of TMS α-helices, was mutated, and the effects on protein subcellular localization and activity assessed. The results showed that the W270A mutant was present exclusively in the ER and formed oligomers with similar activity to those of the wild type HO-1. Interestingly, the W270N mutant was localized not only in the ER, but also in the cytosol and nucleus, suggesting it is susceptible to proteolytic cleavage. Moreover, the microsomal HO activity of the W270N mutant was significantly lower than that of the wild type. The W270N mutation appears to interfere with the oligomeric state, as revealed by a lower FRET efficiency. Collectively, these data suggest that oligomerization, driven by TMS-TMS interactions, is crucial for the stabilization and function of HO-1 in the ER.Heme oxygenase (HO)³ catalyzes the NADPH cytochrome P450 reductase-dependent oxidative degradation of cellular heme to biliverdin, carbon monoxide (CO), and free iron (1, 2). Biliverdin is subsequently converted to bilirubin by biliverdin reductase in the cytosol. Two HO isoforms have been identified in mammalian systems. HO-1 is a 288 amino acid protein and is expressed at high amounts in a variety of pathological conditions associated with cellular stress. There is compelling evidence that HO-1 induction represents an important cytoprotective defense mechanism against oxidative insults by virtue of the anti-oxidant properties of the bilirubin and the anti-inflammatory effect of the CO produced (2). HO-1 is anchored in the endoplasmic reticulum (ER) through a single transmembrane segment (TMS) located at the C terminus, while the rest of the molecule is cytoplasmic (3). HO-1 is sensitive to proteolytic cleavage (4), and it was recently shown that HO-1 can be proteolytically cleaved from the ER and translocated to the nucleus under certain stress conditions (5). Although the catalytic site in the cytoplasmic domain remains intact, the activity of soluble HO-1 is drastically reduced (5), indicating that ER localization is important for its full enzymatic function.Self-assembly to form dimers and higher oligomers is a common phenomenon in many membrane proteins (6, 7). Numerous studies have revealed that interactions between TMSs play an important role in the structure and function of many membrane proteins. Examples include receptors, enzymes, neurotransmitter transporters, and ion channels, in which oligomerization is crucial for their proper cellular localization and function (8). HO-1 does not contain any cysteine residues and has therefore been assumed to function as a monomer (1). To determine whether HO-1 forms oligomers in native membranes, in the present study, we performed chemical cross-linking, co-immunoprecipitation, and FRET analysis using fluorescent protein tags fused to the N terminus of HO-1. The results showed that HO-1 formed dimers/oligomers in the ER and that the TMS provided the interface for the protein-protein interactions. Interference with the TMS-TMS interaction resulted in destabilization of HO-1 and a reduction in enzymatic function.     

A novel method for identifying hydrophobicity on fungal surfaces

Fungal surface hydrophobicity has many ecological functions and water contact angles measurement is a direct and simple approach for its characterization. The objective of this study was to evaluate if in-vitro growth conditions coupled with versatile image analysis allows for more accurate fungal contact angle measurements. Fungal cultures were grown on agar slide media and contact angles were measured utilizing a modified microscope and digital camera setup. Advanced imaging software was adopted for contact angle determination. Contact angles were observed in hydrophobic, hydrophilic and a newly created chronoamphiphilic class containing fungi taxa with changing surface hydrophobicity. Previous methods are unable to detect slight changes in hydrophobicity, which provide vital information of hydrophobicity expression patterns. Our method allows for easy and efficient characterization of hydrophobicity, minimizing disturbance to cultures and quantifying subtle variation in hydrophobicity.     

Translational control by RGS2

The regulator of G protein signaling (RGS) proteins are a family of guanosine triphosphatase (GTPase)–accelerating proteins. We have discovered a novel function for RGS2 in the control of protein synthesis. RGS2 was found to bind to eIF2Bϵ (eukaryotic initiation factor 2B ϵ subunit) and inhibit the translation of messenger RNA (mRNA) into new protein. This effect was not observed for other RGS proteins tested. This novel function of RGS2 is distinct from its ability to regulate G protein–mediated signals and maps to a stretch of 37 amino acid residues within its conserved RGS domain. Moreover, RGS2 was capable of interfering with the eIF2–eIF2B GTPase cycle, which is a requisite step for the initiation of mRNA translation. Collectively, this study has identified a novel role for RGS2 in the control of protein synthesis that is independent of its established RGS domain function.     

Complement 3 deficiency impairs early pregnancy in mice

Human oviductal cells produce complement‐3 (C3) and its derivative, iC3b. These molecules are important in immune responses. Our recent study suggested that iC3b also possessed embryotrophic activity and it stimulates the blastulation and hatching rates of in vitro cultured mouse embryos. The objective is to study the impact of C3 deficiency on early pregnancy in vivo using homozygous C3‐deficient (C3KO) and wild‐type (C3WT) mice. C3 protein was undetectable in the reproductive tissues of C3KO mice. Deficiency in C3 is associated with significantly longer estrous cycle (P = 0.037). No significant difference was found in the ovulation rate, total cell count in blastocysts and implantation rate between the wild‐type and the C3KO mice, though C3KO mice tended to have lower values in the latter two parameters. On day 15 of pregnancy, C3KO mice had fewer conceptus (P < 0.001) and higher resorption rate (P < 0.001) than that of C3WT mice. The fetal and placental weights (P < 0.001) were lower in the C3KO mice. The placenta of C3KO mice had smaller spongiotrophoblast (P = 0.001) and labyrinth (P = 0.037). Deficiency in C3 is associated with mild impairment in early pregnancy including longer estrous cycle and higher resorption rates after implantation. The impairment may be related to compromised placental development leading to under‐developed fetuses. Mol. Reprod. Dev. 76: 647–655, 2009. © 2009 Wiley‐Liss, Inc.     

Adaptive molecular evolution of virulence genes of avian influenza - A virus subtype H5N1: An analysis of host radiation

The phenomenon of host radiation is strongly influenced by the rates of mutation of their virulence genes. We have studied the molecular evolution of virulence genes (HA, NS, PB2) of the Avian Influenza Virus H5N1 from avian to human hosts. We used a site-specific comparison of synonymous (silent) and non-synonymous (amino acid altering) nucleotide substitutions for the three chosen genes in parasite populations from different hosts. Analyses were made using Maximum Likelihood (ML) genealogies for the null and alternate hypothesis based on differential gamma distribution rates. The null hypothesis had a higher rate of substitution and was found to be more suitable for all the studied genes by Likelihood Ratio Test (LRT). The study showed the NS gene to be having the fastest rate of evolution.     

Dammarane-type saponins from Gynostemma pentaphyllum

Dammarane-type saponins, gypenosides VN1–VN7 (1–7), were isolated from the total saponin extract of Gynostemma pentaphyllum aerial parts, with their structures elucidated on the basis of spectroscopic and chemical methods. These compounds showed moderate cytotoxic activity against four human cancer cell lines, A549 (lung), HT-29 (colon), MCF-7 (breast), and SK-OV-3 (ovary), with IC₅₀ values ranging from 19.6 ± 1.1 to 43.1 ± 1.0 μM. Regarding the HL-60 (acute promyelocytic leukemia) cell line, compounds 1, 5, and 6 showed weakly active with IC₅₀ values of 62.8 ± 1.9, 72.6 ± 3.6, and 82.4 ± 3.2 nM, respectively, while 2, 3, 4, and 7 were less active with IC₅₀ values >100 μM.