3'-Fluoro-3'-deoxy analogs of 2-5A 5'-monophosphate: binding to 2-5A-dependent endoribonuclease and susceptibility to (2'-5')phosphodiesterase degradation

Analogs of 2-5A trimer 5'-monophosphate (2'-5')pA3,p5'A2'p5'A2'p5'A containing 9-(3-fluoro-3-deoxy-c-D-xylofuranosyl)adenine (AF) or 3'-fluoro-3'- deoxyadenosine (AF) at different positions of the chain have been synthesized. All of them were compared with (2'-5')pA3 and (2'-5')pA2 (3'dA) by (i) their ability to bind to 2-5A-dependent endoribonuclease(RNase L) of mouse L cells and of rabbit reticulocyte lysates and (ii) their susceptibility to the degradation by the (2'-5')phosphodiesterase activity. The results of this study suggest that the oligonucleotide conformation is important for its biochemical properties.     

Probing of exosites leads to novel inhibitor scaffolds of HCV NS3/4A proteinase

Background

Hepatitis C is a treatment-resistant disease affecting millions of people worldwide. The hepatitis C virus (HCV) genome is a single-stranded RNA molecule. After infection of the host cell, viral RNA is translated into a polyprotein that is cleaved by host and viral proteinases into functional, structural and non-structural, viral proteins. Cleavage of the polyprotein involves the viral NS3/4A proteinase, a proven drug target. HCV mutates as it replicates and, as a result, multiple emerging quasispecies become rapidly resistant to anti-virals, including NS3/4A inhibitors.

Methodology/Principal Findings

To circumvent drug resistance and complement the existing anti-virals, NS3/4A inhibitors, which are additional and distinct from the FDA-approved telaprevir and boceprevir α-ketoamide inhibitors, are required. To test potential new avenues for inhibitor development, we have probed several distinct exosites of NS3/4A which are either outside of or partially overlapping with the active site groove of the proteinase. For this purpose, we employed virtual ligand screening using the 275,000 compound library of the Developmental Therapeutics Program (NCI/NIH) and the X-ray crystal structure of NS3/4A as a ligand source and a target, respectively. As a result, we identified several novel, previously uncharacterized, nanomolar range inhibitory scaffolds, which suppressed of the NS3/4A activity in vitro and replication of a sub-genomic HCV RNA replicon with a luciferase reporter in human hepatocarcinoma cells. The binding sites of these novel inhibitors do not significantly overlap with those of α-ketoamides. As a result, the most common resistant mutations, including V36M, R155K, A156T, D168A and V170A, did not considerably diminish the inhibitory potency of certain novel inhibitor scaffolds we identified.

Conclusions/Significance

Overall, the further optimization of both the in silico strategy and software platform we developed and lead compounds we identified may lead to advances in novel anti-virals.     

A novel method with improved power to detect recombination hotspots from polymorphism data reveals multiple hotspots in human genes

We introduce a new method for detection of recombination hotspots from population genetic data. This method is based on (a) defining an (approximate) penalized likelihood for how recombination rate varies with physical position and (b) maximizing this penalized likelihood over possible sets of recombination hotspots. Simulation results suggest that this is a more powerful method for detection of hotspots than are existing methods. We apply the method to data from 89 genes sequenced in African American and European American populations. We find many genes with multiple hotspots, and some hotspots show evidence of being population-specific. Our results suggest that hotspots are randomly positioned within genes and could be as frequent as one per 30 kb.     

A novel monoclonal antibody DC63 reveals that inhibitor 1 of protein phosphatase 2A is preferentially nuclearly localised in human brain

Abnormal phosphorylation of tau protein represents one of the major candidate pathological mechanisms leading to Alzheimer's disease (AD) and related tauopathies. Altered phosphorylation status of neuronal tau protein may result from upregulation of tau-specific kinases or from inhibition of tau-specific phosphatases. Increased expression of the protein inhibitor 1 of protein phosphatase 2A (I1PP2A) could therefore indirectly regulate the phosphorylation status of tau. As an important step towards elucidation of the role of I1PP2A in the physiology and pathology of tau phosphorylation, we developed a novel monoclonal antibody, DC63, which recognizes I1PP2A. Specificity of the antibody was examined by mass spectrometry and Western blot. This analysis supports the conclusion that the antibody does not recognize any of the other proteins of the 9-member leucine-rich acidic nuclear phosphoprotein family to which I1PP2A belongs. Immunoblot detection revealed that the inhibitor I1PP2A is expressed throughout the brain, including the hippocampus, temporal cortex, parietal cortex, subcortical nuclei and brain stem. The cerebellum displayed significantly higher levels of expression of I1PP2A than was seen elsewhere in the brain. Imunohistochemical analysis of normal human brain showed that I1PP2A is expressed in both neurons and glial cells and that the protein is preferentially localized to the nucleus. We conclude that the novel monoclonal antibody DC63 could be successfully employed as a mass spectrometry-validated molecular probe that may be used for in vitro and in vivo qualitative and quantitative studies of physiological and pathological pathways involving I1PP2A.     

A novel case of ACOX2 deficiency leads to recognition of a third human peroxisomal acyl-CoA oxidase

Peroxisomal acyl-CoA oxidases catalyze the first step of beta-oxidation of a variety of substrates broken down in the peroxisome. These include the CoA-esters of very long-chain fatty acids, branched-chain fatty acids and the C27-bile acid intermediates. In rat, three peroxisomal acyl-CoA oxidases with different substrate specificities are known, whereas in humans it is believed that only two peroxisomal acyl-CoA oxidases are expressed under normal circumstances. Only three patients with ACOX2 deficiency, including two siblings, have been identified so far, showing accumulation of the C27-bile acid intermediates. Here, we performed biochemical studies in material from a novel ACOX2-deficient patient with increased levels of C27-bile acids in plasma, a complete loss of ACOX2 protein expression on immunoblot, but normal pristanic acid oxidation activity in fibroblasts. Since pristanoyl-CoA is presumed to be handled by ACOX2 specifically, these findings prompted us to re-investigate the expression of the human peroxisomal acyl-CoA oxidases. We report for the first time expression of ACOX3 in normal human tissues at the mRNA and protein level. Substrate specificity studies were done for ACOX1, 2 and 3 which revealed that ACOX1 is responsible for the oxidation of straight-chain fatty acids with different chain lengths, ACOX2 is the only human acyl-CoA oxidase involved in bile acid biosynthesis, and both ACOX2 and ACOX3 are involved in the degradation of the branched-chain fatty acids. Our studies provide new insights both into ACOX2 deficiency and into the role of the different acyl-CoA oxidases in peroxisomal metabolism.     

A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site

Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-type spliceosomes. This protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded protein precipitated the 14 kDa protein cross-linked to the branch adenosine, confirming the identity of the p14 cDNA. A combination of immunoblotting, protein microsequencing and immunoprecipitation revealed that p14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. p14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that p14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.     

Dopamine/serotonin receptor ligands. Part 15: Oxygenation of the benz-indolo-azecine LE 300 leads to novel subnanomolar dopamine D1/D5 antagonists

Relying on the high affinities of the benz-indolo-azecine LE 300 (1) and the hydroxylated dibenz-azecine LE 404 (2b) for the D1/D5 receptor subtypes, we synthesized methoxylated, hydroxylated and an indole-N methylated derivatives of 1 (Fig. 1). Hydroxylation of azecine derivatives is beneficial with regard to the affinities and selectivities for all the dopamine receptor subtypes. The 'serotonin-derived' 3-oxygenated target compounds but not the 11-oxygenated analogues were superior to the unsubstituted LE 300. 11-Methoxy-7,14-dimethyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f][3]benzazecine (3e) was found to be the most potent antagonist at D2/D3/D4 and D5 receptor subtypes (Ki for D5 = 0.23 nmol) of all known benz-indolo-azecines.     

ABO exon and intron analysis in individuals with the AweakB phenotype reveals a novel O1v-A2 hybrid allele that causes four missense mutations in the A transferase

Background  

Since the cloning in 1990 of cDNA corresponding to mRNA transcribed at the blood-group ABO locus, polymorphisms due to ethnic and/or phenotypic variations have been reported. Some subgroups have been explained at the molecular level, but unresolved samples are frequently encountered in the reference laboratory.     

A rapid and simple method to determine the specific activities of serotonin, 5-hydroxyindoleacetic acid,and 5-hydroxytryptophan in brain by HPLC with electrochemical detection

The specific activities of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) have been determined in the brain of rats by HPLC using electrochemical detection. The method allows, from a single sample, the simultaneous measurement of all three compounds and collection of each peak for radioactivity determinations. Five male Wistar rats were injected i.v. with 2.0 mCi/kg ofDl-5-hydroxy-[G-³H]tryptophan (2.6 Ci/mmol) and 30 min later the animals were killed by near freezing. Whole brains were removed and homogenized in an acid medium. The content of 5-HTP, 5-HT, and 5-HIAA were determined by HPLC. Each peak of interest was immediately collected after detection in scintillation vials by use of a small dead space detector (TL-9A, Bioanalytical Systems, Inc.). The amounts of radioactivity were determined and specific activities calculated from the results. A second chromatography system (TLC) was used to check the authenticity and purity of compounds separated by the HPLC.     

Mutations induced by 5-formyl-2'-deoxyuridine in Escherichia coli include base substitutions that can arise from mispairs of 5-formyluracil with guanine, cytosine and thymine

5-Formyluracil (5-foU) is a major oxidation product of thymine formed in yields comparable to that of 8-oxoguanine in DNA by ionizing radiation. Whereas the mutagenic effects of 8-oxoguanine are well understood, the investigation of the biological implications of 5-foU has so far been limited. Here we demonstrate that 5-formyl-2'-deoxyuridine (5-fodUrd) supplied to the growth medium of Escherichia coli induces several base substitutions at different frequencies at position 461 in the lacZ gene in the following order: A.T-->G.C>G.C-->A.T>G.C-->T.A>A.T-->T.A>A.T-->C.G. No induction of G.C-->C.G transversions was observed. It is inferred that 5-fodUrd will be incorporated into the DNA during cell growth, forming mispairs with guanine, cytosine and thymine during replication. It, thus, appears that cell growth in the presence of 5-fodUrd may represent a good model for elucidating the cellular effects of 5-foU residues in DNA.     

A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO₂) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP₆ (phytate), its pyrophosphate derivatives InsP₇ and InsP₈, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP₆, InsP₇ and InsP₈ were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO₂ bead purification also enabled us to quantify InsP₆ in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP₆ phosphatase in human plasma, and secondly, InsP₆ is undetectable in either fluid. These observations seriously question reports that InsP₆ is present in human biofluids and the advisability of using InsP₆ as a dietary supplement.