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Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P

In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.

A novel one-pot olefination reaction has been developed, involving the stereoselective formation of (E)-α,β-unsaturated esters/ketones from the reaction of α-bromocarbonyl compounds with aromatic aldehydes. The reactions use a reagent combination of trichlorosilane, and triethylamine and may proceed via the in situ formation of a trichlorosilyl ketene acetal. The general procedure offers key advantages (high conversions, low quantities of organic soluble by-products) over the conventional Wittig reaction.

A weak screening hit with suboptimal physicochemical properties was optimized against PFKFB3 kinase using critical structure-guided insights. The resulting compounds demonstrated high selectivity over related PFKFB isoforms and modulation of the target in a cellular context. A selected example demonstrated exposure in animals following oral dosing. Examples from this series may serve as useful probes to understand the emerging biology of this metabolic target.

The discovery and optimisation of novel, potent and selective small molecule inhibitors of the α-isoform of type III phosphatidylinositol-4-kinase (PI4Kα) are described. Lead compounds show cellular activity consistent with their PI4Kα potency inhibiting the accumulation of IP1 after PDGF stimulation and reducing cellular PIP, PIP2 and PIP3 levels. Hence, these compounds are useful in vitro tools to delineate the complex biological pathways involved in signalling through PI4Kα.

Two series of inhibitors of type III phosphatidylinositol-4-kinase were identified by high throughput screening and optimised to derive probe compounds that independently and selectively inhibit the α- and the β-isoforms with no significant activity towards related kinases in the pathway. In a cellular environment, inhibition of the α- but not the β-subtype led to a reduction in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate concentration, causing inhibition of inositol-1-phosphate formation and inhibition of proliferation in a panel of cancer cell lines.

Antioxidant enzymes are critical in the process of cellular response against reactive species of oxygen. In mammalian cells, catalase and glutathione peroxidase (GPx) are the principal enzymes involved in removing H 2 O 2 . Schistosoma mansoni does not contain catalase and has been found to possess a cytochrome c peroxidase linked activity in its cells. Cytochrome c peroxidase (CcP) is an enzyme isolated and extensively characterized from yeast. Our interest has been to evaluate the role of cytochrome c peroxidase as a novel antioxidant enzyme in Schistosoma mansoni. This initial study deals with basic kinetics of the parasite enzyme and its similarities with the yeast enzyme and investigates its cellular location. Cytochrome c peroxidase and cytochrome c oxidase enzyme activities were measured in crude enzyme extracts of Schistosoma mansoni male and female adult worms. Schistosoma mansoni CcP shares some properties with yeast cytochrome c peroxidase. The enzymatic oxidation of ferrocytochrome c is favoured by an acidic pH. Schistosoma mansoni CcP has significant affinity for cytochrome c from horse heart and from Saccharomyces cerevisae and it is inhibited by ferricytochrome c , potassium cyanide and sodium azide. CcP is located in the mitochondria of Schistosoma mansoni and fractionation with digitonin indicates that the parasite enzyme is located in the intermembrane space. The K m values for hydrogen peroxide for Schistosoma mansoni CcP and glutathione peroxidase using horse heart cytochrome c as the electron donor were found to be 31 ± 4.4 and 8.0 ± 1.7 μM respectively. Although GPx has a greater affinity for H 2 O 2 than CcP, the level of activity of CcP per mg protein is 10 times greater and is indicative of the role of this enzyme as a major antioxidant in Schistosoma mansoni.

Spectacular advances in structural and molecular biology have added support to the 'RNA world' hypothesis, and provide a mandate for chemistry to explain how RNA might have been generated prebiotically on the early earth. Difficulties in achieving a prebiotically plausible synthesis of RNA, however, have led many to ponder the question posed in the title of this paper. Herein, we review recent experimental work on the assembly of potential RNA precursors, focusing on methods for stereoselective C-C bond construction by aldolisation and related processes. This chemistry is presented in the context of a broader picture of the potential constitutional self-assembly of RNA. Finally, the relative accessibility of RNA and alternative nucleic acids is considered.

The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMalpha DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme-product dissociation step. The optimal temperature for trans-cleavage was 70 degrees C. This result was confirmed when both the SMalpha1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMalpha1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMalpha1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.

Treatment of mice with a single dose of oltipraz (OPZ) at 200 mg/kg led to a significant (P less than 0.05) increase in hepatic cytosolic glutathione S-transferase (GST) activity and content. GST activity monitored with 1,2-dichloro-4-nitrobenzene was increased 3.8-fold 3 days after treatment, suggesting the induction of mu class isoenzymes. Ethacrynic acid, a marker for pi class isoforms, showed only a slight increase in GST activity while no induction was observed with cumene hydroperoxide, an indicator for the alpha class. The increase in mu class isoenzymes was further confirmed by separation of the mouse liver affinity purified GST by chromatofocusing and also by resolving the GST subunits by reverse-phase high performance liquid chromatographic procedures. Therefore, OPZ induces mainly the mu class isoenzymes in mouse hepatic tissues.

The activity of glutathione S-transferase (GST) decreased progressively in Schistosoma mansoni from mice treated with oltipraz (OPZ). However, the peroxidase activity of GST (selenium-independent) and selenium-dependent glutathione peroxidase was not affected by OPZ treatment. Purification and quantification of GST from worms after OPZ treatment indicated that the decrease in enzyme activity was greater than could be accounted for by the decrease in GST protein content. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with GST isoenzyme specific antisera revealed a slight decrease in the quantity of both 26 and 28 kDa GSTs. Fractionation of cytosolic GSTs from male S. mansoni by chromatofocusing resolved three major isoenzymes (SmI, II and III) and a minor form which eluted first from the column. SmI, II and III all had a molecular weight of about 28 kDa on SDS-polyacrylamide gel electrophoresis. However, on electrophoresis in the absence of SDS, the three GST forms exhibited different mobilities. The pattern of SmI, II and III was similar in untreated and OPZ-treated worms, but the activities of the isoenzymes from treated worms were lower. The results suggest that OPZ interacts with the GST isoenzymes SmI, II and III in a similar manner; thus, the effects are not isoenzyme specific. Taken together, these results suggest that OPZ and/or its metabolites interact directly with GST resulting in inhibition of activity and reduction in total enzyme protein. This mechanism may be important in the antischistosomal action of OPZ.

Adult worms of Schistosoma mansoni recovered from mice treated with oltipraz (OPZ) showed a significant diminution in their ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan, a measure of parasite viability. Incubation of glutathione S-transferase (GST) from S. mansoni with OPZ resulted in a time- and concentration-dependent inhibition of enzyme activity. RP 36 642 (an inactive oxy-derivative of OPZ) had a minimal effect on the viability of the worms and no effect on GST activity. The structural integrity of OPZ, particularly the thione sulphur, appears to be necessary for expression of the antischistosomal effects of the drug. OPZ-induced inhibition of GST was non-competitive with either reduced glutathione (GSH) or 1-chloro-2,4-dinitrobenzene (CDNB), indicating that the drug is not a substrate for GST-catalysed conjugation reactions. In addition, the inhibition of GST could not be reversed by dialysis or repurification of the enzyme via a GSH-agarose affinity column. The effects of OPZ on GST activity could render the parasite vulnerable to damage by host-derived reactive oxygen species and aldehydic products of lipid peroxidation. The effects of OPZ on GST activity may play a role in the antischistosomal action of OPZ.

The effects of cell-free generated oxidants on migrating and developing stages of Schistosoma mansoni were investigated and the levels of antioxidant enzymes and of glutathione were determined for each stage. Schistosomula and 2-week-old parasites recovered from the livers of infected mice showed similar susceptibility to killing by added hydrogen peroxide and t-butylhydroperoxide. However, when glucose (0.5 mM)-glucose oxidase (2.5 mU ml−1) and xanthine (0.5 mM) or hypoxanthine (0.5 mM)-xanthine oxidase (5.0 mU ml−1) systems were used to generate hydrogen peroxide and oxygen free-radicals, schistosomula were more susceptible to oxidative killing than the 2-week-old parasites. The 4- and 8-week-old worms were more resistant to oxidants than all of the younger stages. High levels of superoxide dismutase (16.2–24.8 U mg−1 protein) were present in all stages. Catalase was not detected. Glutathione peroxidase activity with cumene hydroperoxide as substrate was not detectable in the schistosomula but the activity was present in the 2-week-old parasites. However, hydrogen peroxide-sensitive glutathione peroxidase activity was present in all the stages with a threefold difference in activity between schistosomula and the adult stages. Glutathione-s-transferase activity was significantly lower in the schistosomula, lung stages, and the 2-week-old parasites than in the older stages. Progressive increases in the levels of glutathione reductase and glutathione were also observed with development. The differences in the levels of antioxidants between different stages of development may partly explain the increase in resistance to oxidant-mediated damage as the parasite develops.

In contrast to schistosomula, adult S. mansoni are relatively resistant to oxidant killing in cell free systems, and possess significantly higher levels of oxidant scavengers. In this study, we observed that the adult S. mansoni had a significantly greater capacity to remove hydrogen peroxide than the schistosomula. In addition, they protected a significant proportion of the schistosomula against oxidant killing in vitro . The susceptibility of schistosomula to oxidant killing was inversely dependent on the weight of schistosomules used per assay. However, when the weight of schistosomules and that of adults were equivalent, the adults afforded greater protection from the lethal effects of the oxidants to the schistosomules than the schistosomules could themselves. The protein content of the adults was significantly greater than that of schistosomula of equal wet weight. A significant proportion of the adult worms was rendered susceptible to killing by hydrogen peroxide, when they were exposed to inhibitors of the enzyme cytochrome c peroxidase or glutathione depleting agents. These findings indicate that adult S. mansoni have a greater capacity for oxidant detoxification than the schistosomula, and suggest that cytochrome c peroxidase and glutathione may be important for hydrogen peroxide detoxification in the parasite.

The relative susceptibility of Schistosoma mansoni schistosomula and adults to cell-free oxidant mediated killing has been studied and correlated with levels of oxidant scavengers in the parasite stages. Over 95% of the schistosomula were killed in H 2 O 2 (0.25 m m )) or H 2 O 2 generated by glucose (5.5 m m )-glucose oxidase (5 mU ml −1 ); and in superoxide, H 2 O 2 , and possibly hydroxyl radical and singlet oxygen, generated by xanthine (0.5 m m )- or acetaldehyde (0.5 m m )-xanthine oxidase (10 mU ml −1 ). Under similar conditions less than 2% of the adults were killed. The oxidant scavenger enzymes superoxide dismutase, cytochrome c peroxidase, glutathione peroxidase, glutathione reductase, and the tripeptide glutathione were present in the parasite, but not the enzyme catalase. Enzyme activities and glutathione levels were significantly lower in the schistosomula than adult worms. Juvenile and adult S. mansoni show differences in susceptibility to in vitro oxidant killing that correlate with differences in their levels of endogenous antioxidants.

A decrease in reduced glutathione (GSH) levels in adult Schistosoma mansoni exposed in vitro to the antischistosomal drug oltipraz (OPZ) (20–60 nM) was accompanied by a significant increase in oxidized glutathione (GSSG) levels. The total glutathione (GSH + GSSG) levels also diminished in drug-treated parasites. The activities of the parasite glutathione peroxidase (GPO), utilizing cumene hydroperoxide as a substrate, and glutathione 5-transferase (GST), measured 18 hr after in vitro incubation with the drug, were elevated significantly, but there were no significant alterations in the activities of the GPO, utilizing H2O2, or glutathione reductase (GR). Drug-treated worms showed increased lipid peroxidation. In vivo, the proportion of the worms recovered from infected mice given OPZ (100 mg/kg body wt) gradually declined with time, to about 30% of that recovered from infected untreated control mice by day 14 after drug administration, and consisted predominantly of male worms. Accompanying this significant decline in the proportion of worms recovered were significant decreases in the activities of the enzymes GR and GST in drug-exposed worms. On the other hand, a slight initial increase in the GPO activity with cumene hydroperoxide was followed by a return to control values, and the GPO activity with H2O2 was decreased only slightly with time. Interestingly, the 4-hydroxyalk-2-enal aldehydes, known products of lipid peroxidation, inhibited the GST reaction with 1-chloro-2,4-dinitrobenzene (CDNB). The OPZ-induced changes in S. mansoni could increase parasite susceptibility to oxidative attack by host phagocytes, and are probably linked with the antischistosomal action of the drug in vivo.

The nucleotide sequences of a 1279-bp sheep elastin cDNA clone, pcSEL1 [ Yoon et al. (1984) Biochem. Biophys. Res. Commun.118, 261–269], and a 1230-bp sheep elastin genomic subclone, pSS1 [ Davidson et al (1984) Biochem. J.220, 643–652], corresponding to a portion of the cDNA clone, were determined. These analyses permitted determination of the 100 amino acids at the carboxy terminus of sheep tropoelastin. A portion of this sequence showed strong homology to known sequences of pig tropoelastin, but most of the sequence had not been previously determined through protein sequencing. Novel aspects of the tropoelastin molecule which have been revealed by the present analyses are (i) the presence of an unusual sequence, KPPKP, which may contribute to crosslink formation; and (ii) the finding of cysteine within a sequence, CLGKSCGRKRK, at the putative carboxy terminus of tropoelastin. Because of the presence of these sequences, it is speculated that the carboxy-terminal region may be of importance in crosslinking tropoelastin molecules to themselves or to other matrix macromolecules. The nucleotide analyses revealed that sheep elastin mRNA contains a 974-bp untranslated sequence at the 3′ end, which appears to be strongly conserved among Species.