Determination of the effect of JO146, a CtHtrA inhibitor, on the chlamydial developmental cycle and persistence

The role of Chlamydia trachomatis HtrA (CtHtrA) for the growth and pathogenesis of this organism is presented. Inhibition of CtHtrA led to loss of infectious progeny in Chlamydia, particularly during heat stress or recovery from penicillin persistence. Isolation of CtHtrA inhibitor resistant mutants identified positive selection for mutants in fatty acid related pathways. Importantly, HtrA inhibition was effective against clinical isolates of C. trachomatis. Thus the findings combined indicate that CtHtrA is an important chlamydial growth factor that has the potential to be targeted for the development of anti-chlamydial drugs in the future.

The complementation of yeast with human or Plasmodium falciparum Hsp90 confers differential inhibitor sensitivities

Developing novel drugs against the unicellular parasite Plasmodium is complicated by the paucity of simple screening systems. Heat-shock proteins are an essential class of proteins for the parasite's cyclical life style between different cellular milieus and temperatures. The molecular chaperone Hsp90 assists a large variety of proteins, but its supporting functions for many proteins that are important for cancer have made it into a well-studied drug target. With a better understanding of the differences between Hsp90 of the malarial parasite and Hsp90 of its human host, new therapeutic options might become available. We have generated a set of isogenic strains of the budding yeast Saccharomyces cerevisiae where the essential yeast Hsp90 proteins have been replaced with either of the two human cytosolic isoforms Hsp90 alpha or Hsp90 beta, or with Hsp90 from Plasmodium falciparum (Pf). All strains express large amounts of the Flag-tagged Hsp90 proteins and are viable. Even though the strain with Pf Hsp90 grows more poorly, it provides a tool to reconstitute additional aspects of the parasite Hsp90 complex and its interactions with substrates in yeast as a living test tube. Upon exposure of the set of Hsp90 test strains to the two Hsp90 inhibitors radicicol (Rd) and geldanamycin (GA), we found that the strain with Pf Hsp90 is relatively more sensitive to GA than to Rd compared to the strains with human Hsp90's. This indicates that this set of yeast strains could be used to screen for new Pf Hsp90 inhibitors with a wider therapeutic window.

N-[2-naphthyl]-glycine hydrazide, a potent inhibitor of dna-dependent rna-polymerase of mycobacterium-tuberculosis h37rv

N-[2-Naphthyl]-glycine hydrazide has been shown for the first time as a potent inhibitor of the DNA-dependent RNA polymerase (EC 2.7.7.6) of Mycobacterium tuberculosis H37Rv. At a concentration of 10 to the power -9 M, the compound shows maximum inhibition of the enzyme, the inhibition being less at higher concentrations. It is suggested that the novel type of inhibition pattern may be due to hydrophobic interactions occurring between the molecules of the compound at higher concentrations. The finding that there is a shift in the max of the compound could also account for this phenomenon. The effect of this compound was also tested on DNA-dependent RNA polymerases from an eukaryotic fungus, Microsporum canis. At a concentration of 10 to the power-9 M it inhibits RNA polymerase II (32 percent) but not RNA polymerases I and III.

A concise synthesis of the bioactive meroterpenoid natural product (+/-)-liphagal, a potent PI3K inhibitor

A short, diversity-oriented synthesis that follows a biomimetic route to the marine natural product liphagal, from a commercially available building block, is delineated.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Influence of different nitrogen rates and DMPP nitrification inhibitor on annual N2/O emissions from a subtropical wheat–maize cropping system

Global cereal production will need to increase by 50% to 70% to feed a world population of about 9 billion by 2050. This intensification is forecast to occur mostly in subtropical regions, where warm and humid conditions can promote high N2O losses from cropped soils. To secure high crop production without exacerbating N2O emissions, new nitrogen (N) fertiliser management strategies are necessary. This one-year study evaluated the efficacy of a nitrification inhibitor (3,4-dimethylpyrazole phosphate—DMPP) and different N fertiliser rates to reduce N2O emissions in a wheat–maize rotation in subtropical Australia. Annual N2O emissions were monitored using a fully automated greenhouse gas measuring system. Four treatments were fertilized with different rates of urea, including a control (40 kg-N ha−1 year−1), a conventional N fertiliser rate adjusted on estimated residual soil N (120 kg-N ha−1 year−1), a conventional N fertiliser rate (240 kg-N ha−1 year−1) and a conventional N fertiliser rate (240 kg-N ha−1 year−1) with nitrification inhibitor (DMPP) applied at top dressing. The maize season was by far the main contributor to annual N2O emissions due to the high soil moisture and temperature conditions, as well as the elevated N rates applied. Annual N2O emissions in the four treatments amounted to 0.49, 0.84, 2.02 and 0.74 kg N2O–N ha−1 year−1, respectively, and corresponded to emission factors of 0.29%, 0.39%, 0.69% and 0.16% of total N applied. Halving the annual conventional N fertiliser rate in the adjusted N treatment led to N2O emissions comparable to the DMPP treatment but extensively penalised maize yield. The application of DMPP produced a significant reduction in N2O emissions only in the maize season. The use of DMPP with urea at the conventional N rate reduced annual N2O emissions by more than 60% but did not affect crop yields. The results of this study indicate that: (i) future strategies aimed at securing subtropical cereal production without increasing N2O emissions should focus on the fertilisation of the summer crop; (ii) adjusting conventional N fertiliser rates on estimated residual soil N is an effective practice to reduce N2O emissions but can lead to substantial yield losses if the residual soil N is not assessed correctly; (iii) the application of DMPP is a feasible strategy to reduce annual N2O emissions from sub-tropical wheat–maize rotations. However, at the N rates tested in this study DMPP urea did not increase crop yields, making it impossible to recoup extra costs associated with this fertiliser. The findings of this study will support farmers and policy makers to define effective fertilisation strategies to reduce N2O emissions from subtropical cereal cropping systems while maintaining high crop productivity. More research is needed to assess the use of DMPP urea in terms of reducing conventional N fertiliser rates and subsequently enable a decrease of fertilisation costs and a further abatement of fertiliser-induced N2O emissions.

Theoretical studies on penicillins, penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues: binding specificities of transeptidases and penicillinases

Empirical potential energy calculations have been carried out to determine the preferred conformations of penicillins and penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues. With the exception of 1-oxa-1-dethia penicillins, all the other compounds favour C2 and the C3 puckered conformations of their five-membered rings. Replacement of C2 methyl groups by hydrogen atoms as in bisnorpenicillin V or oxidation of sulphur in position 1 as in sulphones, makes the C3 puckered form much less favourable. Addition of an amino-acyl group at the C6 atom, however, makes the C3 puckered form more favoured in penicillin G or V and in 1-carba-1-dethia penicillins. Through the replacement of the sulphur atom at position 1 by an oxygen atom or by a -CH2 group increases the non-planarity of the lactam peptide bond, it significantly affects the relative disposition of the C3 carboxyl group with respect to the β-lactam ring. These conformational differences have been correlated with the biological activities of these compounds. The present study suggests that the conformation of the bicyclic ring system may be more important for initial binding with the crosslinking enzyme(s) involved in the biosynthesis of bacterial cell-wall peptidoglycan and that the mode of binding is influenced by the nature of the side-group at the C6 atom. These studies predict, in agreement with experimental results, that the 1-oxa-1-dethia penicillin nulceus is an inhibitor of penicillianses. The study also suggests that the stereospecificities of the crosslinking enzyme(s) and penicillinases are very similar with regard to the nature of the side-group at the 6 atom and the confirmation of the bicyclic ring system. However, the confirmational requirement for the bicyclic ring system appears to be more specific in the former enzyme than in the latter.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Rhipicephalus microplus serine protease inhibitor family: annotation, expression and functional characterisation assessment

Background: Rhipicephalus (Boophilus) microplus evades the host's haemostatic system through a complex protein array secreted into tick saliva. Serine protease inhibitors (serpins) conform an important component of saliva which are represented by a large protease inhibitor family in Ixodidae. These secreted and non-secreted inhibitors modulate diverse and essential proteases involved in different physiological processes. Methods: The identification of R. microplus serpin sequences was performed through a web-based bioinformatics environment called Yabi. The database search was conducted on BmiGi V1, BmiGi V2.1, five SSH libraries, Australian tick transcriptome libraries and RmiTR V1 using bioinformatics methods. Semi quantitative PCR was carried out using different adult tissues and tick development stages. The cDNA of four identified R. microplus serpins were cloned and expressed in Pichia pastoris in order to determine biological targets of these serpins utilising protease inhibition assays. Results: A total of four out of twenty-two serpins identified in our analysis are new R. microplus serpins which were named as RmS-19 to RmS-22. The analyses of DNA and predicted amino acid sequences showed high conservation of the R. microplus serpin sequences. The expression data suggested ubiquitous expression of RmS except for RmS-6 and RmS-14 that were expressed only in nymphs and adult female ovaries, respectively. RmS-19, and -20 were expressed in all tissues samples analysed showing their important role in both parasitic and non-parasitic stages of R. microplus development. RmS-21 was not detected in ovaries and RmS-22 was not identified in ovary and nymph samples but were expressed in the rest of the samples analysed. A total of four expressed recombinant serpins showed protease specific inhibition for Chymotrypsin (RmS-1 and RmS-6), Chymotrypsin / Elastase (RmS-3) and Thrombin (RmS-15). Conclusion: This study constitutes an important contribution and improvement to the knowledge about the physiologic role of R. microplus serpins during the host-tick interaction.

Purification and Regulatory Properties of Mung Bean (Vigna Radiata L.) Serine Hydroxymethyltransferase 1

Serine hydroxymethyltransferase, the first enzyme in the pathway for interconversion of C1 fragments, was purified to homogeneity for the first time from any plant source. The enzyme from 72-h mung bean (Vigna radiata L.) seedlings was isolated using Blue Sepharose CL-6B and folate-AH-Sepharose-4B affinity matrices and had the highest specific activity (1.33 micromoles of HCHO formed per minute per milligram protein) reported hitherto. The enzyme preparation was extremely stable in the presence of folate or L-serine. Pyridoxal 5'-phosphate, ethylenediaminetetraacetate and 2-mercaptoethanol prevented the inactivation of the enzyme during purification. The enzyme functioned optimally at pH 8.5 and had two temperature maxima at 35 and 55°C. The Km values for serine were 1.25 and 68 millimolar, corresponding to Vmax values of 1.8 and 5.4 micromoles of HCHO formed per minute per milligram protein, respectively. The K0.5 value for L-tetrahydrofolate (H4folate) was 0.98 millimolar. Glycine, the product of the reaction and D-cycloserine, a structural analog of D-alanine, were linear competitive inhibitors with respect to L-serine with Ki values of 2.30 and 2.02 millimolar, respectively. Dichloromethotrexate, a substrate analog of H4folate was a competitive inhibitor when H4folate was the varied substrate. Results presented in this paper suggested that pyridoxal 5'-phosphate may not be essential for catalysis.The sigmoid saturation pattern of H4folate (nH = 2.0), one of the substrates, the abolition of sigmoidicity by NADH, an allosteric positive effector (nH = 1.0) and the increase in sigmoidicity by NAD+ and adenine nucleotides, negative allosteric effectors (nH = 2.4) clearly established that this key enzyme in the folate metabolism was an allosteric protein. Further support for this conclusion were the observations that (a) serine saturation exhibited an intermediary plateau region; (b) partial inhibition by methotrexate, aminopterin, O-phosphoserine, DL-{alpha}-methylserine and DL-O-methylserine; (c) subunit nature of the enzyme; and (d) decrease in the nH value from 2.0 for H4folate to 1.5 in presence of L-serine. These results highlight the regulatory nature of mung bean serine hydroxymethyltransferase and its possible involvement in the modulation of the interconversion of folate coenzymes.

Impact of 1-methylcyclopropene treatment on the sensory quality of 'Bartlett' pear fruit

The ethylene biosynthesis inhibitor, 1-methylcyclopropene (1-MCP), has been commercially used to extend the storage life of European pear fruit and to allow shipment to distant markets. However, the influence of 1-MCP on the ability of fruit to ripen to an acceptable sensory quality has not been investigated in 'Bartlett' pear, one of the most aromatic of pear fruit. In the current study, early-, mid- and late-season 'Bartlett' pear fruit were treated with 0.6μLL-1 1-MCP at 0°C for 24h, 100μLL-1 ethylene at 20°C for 24h, or untreated before immediate transfer to 20°C for ripening until eating soft (13N firmness). Fruit from each treatment were subjected to objective and sensory quality evaluation once ripe. Sensory quality showed differences related to harvest maturity; the importance of harvest maturity depended on the treatment. Treatment with 1-MCP effectively slowed fruit ripening and therefore extended postharvest life, but also resulted in fully ripened pears with more desirable sensory traits, as compared with ethylene-treated and untreated pears. The enhanced sensory quality was related to higher sweetness, juiciness and pear aroma and reduced fermented aroma, gritty texture and tart taste. In addition, the sensory profiles of 1-MCP-treated pears were more stable among fruit from the three harvest dates than the ethylene-treated pears, which exhibited very different sensory profiles between early- and late-season fruits. © 2015 Elsevier B.V.

Annual nitrogen dynamics and urea fertilizer recoveries from a dairy pasture using 15N; effect of nitrification inhibitor DMPP and reduced application rates

Direct nitrogen (N) losses from pastures contribute to the poor nitrogen use efficiency of the dairy industry, though the exact fate of applied N and the processes involved are largely unknown. Nitrification inhibitors such as DMPP can potentially increase fertilizer N use efficiency (NUE), though few studies globally have examined the effectiveness of DMPP coated urea in pastures. This study quantified the NUE of DMPP combined with reduced application rates, and the effect on N dynamics and plant–soil interactions over an annual ryegrass/kikuyu rotation in Queensland, Australia. Labeled 15N urea and DMPP was applied over 7 winter applications at standard farmer (45 kg N ha−1) and half (23 kg N ha−1) rates. Fertilizer recoveries and NUE were calculated over 13 harvests, and the contribution of fertilizer and soil N estimated. Up to 85% of the annual N harvested was from soil organic matter. DMPP at the lower rate increased annual yields by 31% compared to the equivalent urea treatment with no difference to the high N rates. Almost 40% of the N added at the conventional fertilizer application rate as urea was lost to the environment; 80 kg N ha−1 higher than the low DMPP. Combining the nitrification inhibitor DMPP with reduced fertilizer application rates shows substantial potential to reduce N losses to the environment while sustaining productivity in subtropical dairy pastures.

Structure-Based Design of DevR Inhibitor Active against Nonreplicating Mycobacterium tuberculosis

Antitubercular treatment is directed against actively replicating organisms. There is an urgent need to develop drugs targeting persistent subpopulations of Mycobacterium tuberculosis. The DevR response regulator is believed to play a key role in bacterial dormancy adaptation during hypoxia. We developed a homology-based model of DevR and used it for the rational design of inhibitors. A phenylcoumarin derivative (compound 10) identified by in silico pharmacophore-based screening of 2.5 million compounds employing protocols with some novel features including a water-based pharmacophore query, was characterized further. Compound 10 inhibited DevR binding to target DNA, down-regulated dormancy genes transcription, and drastically reduced survival of hypoxic but not nutrient-starved dormant bacteria or actively growing organ ` isms. Our findings suggest that compound 10 ``locks'' DevR in an inactive conformation that is unable to bind cognate DNA and induce the dormancy regulon. These results provide proof-of-concept for DevR as a novel target to develop molecules with sterilizing activity against tubercle bacilli.

Blockade of estrogen synthesis with an aromatase inhibitor affects luteal function of the pseudopregnant rat

The luteotropic action of estrogen (E) was investigated using immature pseudopregnant rat as the model and CGS 16949A (Fadrozole hydrochloride), a potent aromatase inhibitor (AI), to block E synthesis. Aromatase activity could be inhibited by administering CGS 16949A (50 mu g/day/rat) via a mini osmotic Alzet pump (model 2002) for 3 days during pseudopregnancy. This resulted in significant reduction of serum (40%, P < 0.05) and intraovarian (70.6%, P < 0.001) estradiol-17 beta (E(2)) levels. The serum and intraovarian progesterone (P-4) levels as analyzed on day 4 of pseudopregnancy were also reduced by greater than or equal to 50% (for both, P < 0.01). Simultaneous administration of estradiol-3-benzoate (E(2)B) via an Alzet pump during the Al: treatment period at a dose of 1 mu g/day could completely reverse the Al induced reduction in P-4 secretion. The luteal cells of experimental rats depleted of E in vivo showed a significantly reduced response upon incubation with hCG or dbcAMP in vitro (P < 0.05 and 0.001, respectively). Addition of E(2) (500 pg/tube) at the time of in vitro incubation was able to partially increase the responsiveness to hCG. The luteal cell LH/hCG receptor content and the affinity of hCG binding to the receptor remained unchanged following AI treatment in vivo. Both esterified and total cholesterol content of luteal cells of rats treated with Al in vivo was significantly high (P < 0.05) suggesting that E lack results in an impairment in cholesterol utilization for steroidogenesis. The results clearly show that E regulates luteal function in the pseudopregnant rat by acting at a non-cAMP mediated event and this perhaps involves facilitation of cholesterol utilization at the mitochondrial level for P-4 synthesis.