Chemical profiling of deoxyhypusine hydroxylase inhibitors for antimalarial therapy

A first approach to discover new antimalarials has been recently performed in a combined approach with data from GlaxoSmithKline Tres Cantos Antimalarial Set, Novartis-GNF Malaria Box Data set and St. Jude Children’s Research Hospital. These data are assembled in the Malaria Box. In a first phenotypic forward chemical genetic approach, 400 chemicals were employed to eradicate the parasite in the erythrocytic stages. The advantage of phenotypic screens for the identification of novel chemotypes is that no a priori assumptions are made concerning a fixed target and that active compounds inherently have cellular bioavailability. In a first screen 40 mostly heterocyclic, highly active compounds (in nmol range of growth inhibition) were identified with EC₅₀ values ≤2 μM against chloroquine-resistant Plasmodium falciparum strains and a therapeutic window ≥10 against two mammalian cell lines. 78 % of the compounds had no violations with the Lipinski Rule of 5 and only 1 % of the compounds showed cytotoxicity when applied at concentrations of 10 μM. This pre-selective step of parasitic eradication will be used further for a test of the Malaria Box with a potential in iron chelating capacity to inhibit deoxyhypusine hydroxylase (DOHH) from P. falciparum and vivax. DOHH, a metalloprotein which consists of ferrous iron and catalyzes the second step of the posttranslational modification at a specific lysine in eukaryotic initiation factor 5A (EIF-5A) to hypusine. Hypusine is a novel, non-proteinogenic amino acid, which is essential in eukaryotes and for parasitic proliferation. DOHH seems to be a “druggable” target, since it has only 26 % amino acid identity to its human orthologue. For a High-throughput Screening (HTS) of DOOH inhibitors, rapid and robust analytical tools are a prerequisite. A proteomic platform for the detection of hypusine metabolites is currently established. Ultra performance Liquid Chromatography enables the detection of hypusine metabolites with retention times of 7.4 min for deoxyhypusine and 7.3 min for hypusine. Alternatively, the analytes can be detected by their masses with gas chromatography/mass spectrometry or one-dimensional chromatography coupled to mass spectrometry. Moreover, the identified hits will be tracked further to test their efficacy in novel “in vitro assays”. Subsequently in vivo inhibition in a humanized mouse model will be tested.     

Evidence for conformational changes in the yeast deoxyhypusine hydroxylase Lia1 upon iron displacement from its active site

The unique amino acid hypusine is formed exclusively in eIF5A by the successive action of deoxyhypusine synthase and deoxyhypusine hydroxylase (yeast Lia1, human DOHH). Although the first enzyme has been extensively studied, both Lia1 structure and the mechanism of action remain unclear. Hence, a multi-approach was used to evaluate Lia1 catalysis, metal/substrate binding, structural conformation and stability. Mutational analyses of Lia1 revealed fine differences in the mode of substrate binding between the human and yeast counterparts. Like human DOHH, recombinant Lia1 is an iron metalloenzyme. Iron is essential for enzyme activity since its loss renders the enzyme totally inactive. The separation of iron-free and iron-bound forms by gel filtration and native electrophoresis suggests differences in Lia1 tertiary structure related to the iron binding. The ability of Lia1 to undergo conformational changes prompted us to use a set of complementary spectroscopic approaches and SAXS to obtain detailed information on the processes underlying dissociation of iron from Lia1 at different levels of the protein organization. The additive effect of weak interactions, especially within the metal center, resulted in an active enzyme in a stabilized and compact three-dimensional fold. Loss of tertiary contacts upon iron displacement led to an elongated conformation of Lia1, in which the N- and C-terminal domains are no longer in close proximity to guarantee the proper orientation of the active groups within the active site pocket. Our results demonstrate an essential structural role for iron binding in addition to its contribution to the catalysis of hypusine formation in the eIF-5A precursor.     

Molecular cloning and functional expression of bovine deoxyhypusine hydroxylase cDNA and homologs

Deoxyhypusine hydroxylase is the second of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here, we describe the cloning and characterization of bovine deoxyhypusine hydroxylase cDNA and its homologs. The deduced bovine deoxyhypusine hydroxylase protein is 87% identical to human enzyme and 45% identical to yeast enzyme. The overexpressed enzyme showed activity in catalyzing the hydroxylation of the deoxyhypusine residue in the eIF5A intermediate. An amino acid substitution from Glu 57 to Gly located at one of the four conserved His-Glu (HE) pairs, the potential metal coordination sites, resulted in severe reduction of deoxyhypusine hydroxylase activity. A deletion at the HEAT-repeats 1-3 resulted in complete losses of deoxyhypusine hydroxylase activity.     

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on the enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion bunding capacity of the chelators.     

Specific assays for bacteria using phage mediated release of adenylate kinase

A sensitive and rapid assay method for the specific detection of bacteria was developed using Escherichia coli and Salmonella newport as the test organisms. Bacteriophages were used to provide specific lysis of the bacteria and then the release of cell contents was measured by ATP bioluminescence. Increased sensitivity was obtained by focusing on the bacteria's adenylate kinase (AK) as the cell marker instead of ATP as conventionally used. Fewer than 10³ E. coli cells could be readily detected in less than 1 h. Salmonella newport assays, although as sensitive, were slower and took up to 2 h. The effects of the culture medium, the phage, and the presence of non-specific bacteria were examined.     

The Drosophila deoxyhypusine hydroxylase homologue nero and its target eIF5A are required for cell growth and the regulation of autophagy

Hypusination is a unique posttranslational modification by which lysine is transformed into the atypical amino acid hypusine. eIF5A (eukaryotic initiation factor 5A) is the only known protein to contain hypusine. In this study, we describe the identification and characterization of nero, the Drosophila melanogaster deoxyhypusine hydroxylase (DOHH) homologue. nero mutations affect cell and organ size, bromodeoxyuridine incorporation, and autophagy. Knockdown of the hypusination target eIF5A via RNA interference causes phenotypes similar to nero mutations. However, loss of nero appears to cause milder phenotypes than loss of eIF5A. This is partially explained through a potential compensatory mechanism by which nero mutant cells up-regulate eIF5A levels. The failure of eIF5A up-regulation to rescue nero mutant phenotypes suggests that hypusination is required for eIF5A function. Furthermore, expression of enzymatically impaired forms of DOHH fails to rescue nero clones, indicating that hypusination activity is important for nero function. Our data also indicate that nero and eIF5A are required for cell growth and affect autophagy and protein synthesis.     

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses L-[carboxy-14C]tyrosine as the substrate, 14CO2 is released from the products of the hydroxylation and further metabolism of L-[carboxy-14C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.     

Investigation of methods for measurement of radioactivity in tritiated DNA and applications to assays for DNA polymerase activity

Methods for collection and counting of ³H-labeled DNA on nitrocellulose and glass fiber filters have been investigated. The findings are of potential importance in determining the amount of radioactivity in DNA as well as other macromolecules. The highest counting efficiencies were observed using glass fiber filters, NCS for dissolving DNA, and a toluene seintillation mixture for counting. However, glass fiber filters, even with large amounts of co-precipitant albumin, failed to collect all of low molecular weight (approx 185,000 daltons) DNA. Thus, in many applications nitrocellulose filters proved to be more advantageous.     

Kinetic and isotopic studies of the oxidative half-reaction of phenol hydroxylase

Phenol hydroxylase, an FAD-containing monooxygenase, catalyzes the conversion of substituted phenols to the corresponding catechol. Use of metapyrocatechase, capable of dioxygenation of several catechols to give highly absorbing products, permitted determination of the time course of product release from phenol hydroxylase. Product dissociated prior to complete reoxidation of the enzyme, most likely concomitant with formation of the 4a-hydroxyflavin species (intermediate III). Deuterated phenol and thiophenol exhibited no kinetic isotope effect during the oxidative half-reaction. Isotope effects of 1.7 to 3.7 were found with resorcinol for the conversion of the second intermediate to intermediate III. These effects limited the possible models for phenol hydroxylation. An attempt was made to distinguish whether the spectrum of intermediate II is due entirely to that of the flavin moiety of phenol hydroxylase or whether some radical intermediate form involved in the formation of catechol makes a significant visible contribution. Reduced native and 6-hydroxy-FAD phenol hydroxylase were reacted with oxygen and resorcinol in order to provide evidence for the identity of intermediate II.     

Effect of substrate and pH on the oxidative half-reaction of phenol hydroxylase

The oxidative half-reaction of phenol hydroxylase has been studied by stopped-flow spectrophotometry. Three flavin-oxygen intermediates can be detected when the substrate is thiophenol, or m-NH2, m-OH, m-CH3, m-Cl, or p-OH phenol. Intermediate I, the flavin C(4a)-hydroperoxide, has an absorbance maximum at 380-390 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate III, the flavin C(4a)-hydroxide, has an absorbance maximum at 365-375 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate II has absorbance maxima of 350-390 nm and extinction coefficients of 10,000-16,000 M-1 cm-1 depending on the substrate. A Hammett plot of the logarithm of the rates of the oxygen transfer step, the conversion of intermediate I to intermediate II, gives a straight line with a slope -0.5. Fluoride ion is a product of the enzymatic reaction when 2,3,5,6-tetrafluorophenol is the substrate. These results are consistent with an electrophilic substitution mechanism for oxygen transfer. The conversions of I to II and II to III are acid-catalyzed. A kinetic isotope effect of 8 was measured for the conversion of II to III using deuterated resorcinol as substrate. The conversion of III to oxidized enzyme is base-catalyzed, suggesting that the reaction depends on the removal of the flavin N(5) proton. Product release occurs at the same time as the formation of intermediate III, or rapidly thereafter. The results are interpreted according to the ring-opened model of Entsch et al. (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563).     

Development and evaluation of yeast-based GFP and luciferase reporter assays for chemical-induced genotoxicity and oxidative damage

Applied Microbiology and Biotechnology - We aimed to develop the bioassays for genotixicity and/or oxidative damage using the recombinant yeast. A genotoxicity assay was developed using recombinant...     

Interferon-gamma release assays for the diagnosis of extrapulmonary tuberculosis: a systematic review and meta-analysis

Interferon -gamma release assays (IGRAs) provide a new diagnostic method for Mycobacterium tuberculosis (TB) infection. However, the diagnostic value of IGRAs for extrapulmonary TB (EPTB) has not been clarified. We searched several databases and selected papers with strict inclusion criteria, evaluated the evidence of commercially available IGRAs (QuantiFERON(?) -TB Gold QFT-G or QFT-GIT and T-SPOT(?) .TB) on blood and the tuberculin skin test (TST) using random effects models. Twenty studies with 1711 patients were included. After excluding indeterminate results, pooled sensitivity for the diagnosis of EPTB was 72% [95% confidence interval (CI) 65-79%] for QFT-G or GIT and 90% (95% CI, 86-93%) for T-SPOT; in high-income countries the sensitivity of QFT-G or GIT (79%, 95% CI 72-86%) was much higher than that (29%, 95% CI 14-48%) in low/middle-income countries. Pooled specificity for EPTB was 82% (95% CI 78-87%) for QFT-G or GIT and 68% (95% CI 64-73%) for T-SPOT. Pooled sensitivity of TST from four studies in high-income countries was lower than that of IGRAs. T-SPOT was more sensitive in detecting EPTB than QFT-G or GIT and TST. However, both IGRAs and TST have similar specificity for EPTB. IGRAs have limited value as diagnostic tools to screen and rule out EPTB, especially in low/middle-income countries. The immune status of patients does not affect the diagnostic accuracy of IGRAs for EPTB.     

A paradigm for determining the relevance of short-term assays: application to oxidative mutagenesis

A simple substructure-based approach was developed to determine whether a short-term assay under development is related mechanistically to the endpoint it seeks to predict. Thus, substructures associated with mutagenicity in Salmonella are also present in carcinogens and agents active in other mutagenicity and genotoxicity assays.When applied to test results obtained with an Escherichia coli strain designed to identify oxidative mutagens, there was no significant association with either carcinogens or mutagens and genotoxicants detected by other systems. There was, however, a significant association between alerts for oxidative mutagenesis and chemicals capable of inducing allergic contact dermatitis (ACD) in humans.     

Chemiluminescent assays for choline and phospholipase-D using a flow injection system

A flow injection chemiluminescent method is described for the determination of choline. The method is based on the production of hydrogen peroxide from choline using on-line covalently bound immobilized choline oxidase column. The product is mixed downstream and detected via the cobalt catalyzed chemiluminescent oxidation of luminol. The detection limit is 1×10⁻⁷ mol/L, with rsd 1.8 to 2.8% in the range 2–10×10⁻⁵ mol/L. The sample throughput is 30 per hour. The method was applied to the determination of choline produced off-line from phosphatidylcholine using phospholipase-D isolated from cabbage. © 1997 John Wiley & Sons, Ltd.     

Simultaneous detection of Human Immunodeficiency Virus 1 and Hepatitis B virus infections using a dual-label time-resolved fluorometric assay

A highly specific and novel dual-label time-resolved immunofluorometric assay was developed exploiting the unique emission wavelengths of the intrinsically fluorescent terbium (Tb³⁺) and europium (Eu³⁺) tracers for the simultaneous detection of human immunodeficiency virus 1 (HIV-1) and hepatitis B virus (HBV) infections, respectively. HIV-1 infection was detected using a double antigen sandwich format wherein anti-HIV-1 antibodies were captured using an in vivo biotinylated version of a chimeric HIV-1 antigen and revealed using the same antigen labeled with Tb³⁺ chelate. Hepatitis B surface antigen (HBsAg), which served as the marker of HBV infection, was detected in a double antibody sandwich using two monoclonal antibodies (mAbs), one chemically biotinylated to capture, and the other labeled with Eu³⁺ nanoparticles, to reveal. The performance of the assay was evaluated using a collection (n = 60) of in-house and commercially available human sera panels. This evaluation showed the dual-label assay to possess high degrees of specificity and sensitivity, comparable to those of commercially available, single analyte-specific kits for the detection of HBsAg antigen and anti-HIV antibodies. This work demonstrates the feasibility of developing a potentially time- and resource-saving multiplex assay for screening serum samples for multiple infections in a blood bank setting.