Journal: Febs Letters

Article Title: 4‐nitrobenzoate inhibits 4‐hydroxybenzoate polyprenyltransferase in malaria parasites and enhances atovaquone efficacy

doi: 10.1002/1873-3468.70186

Figure Lengend Snippet: Ubiquinone biosynthesis pathway. Schematic representation of the ubiquinone biosynthetic pathway (brown), showing the chemical structures of precursors, intermediates, and UQ itself. The enzyme 4‐HPT (EC 2.5.1.39), the hypothetical target of 4‐NB in malaria parasites, is inside a brown box. Discontinuous lines represent multiple steps; red lines indicate drug inhibition. The chemical structure of 4‐NB is boxed, to indicate its inhibitor effect. The figure also shows UQ redox recycling in the mitochondrial electron transport chain (mtETC, green) and the chemical structure and molecular target of atovaquone. Abbreviations in the figure: Ubiquinone (UQ‐n) is the oxidized form of coenzyme Q with an isoprenoid side chain of length n , while ubiquinol (UQH2‐n) is its reduced form; Pf COQ2 refers to the P. falciparum 4‐hydroxybenzoate polyprenyltransferase enzyme; 4‐hydroxybenzoic acid (4‐HB) is a natural precursor of UQ, and 4‐nitrobenzoic acid (4‐NB) is a synthetic analog used as an inhibitor; dihydroorotate dehydrogenase (DHODH) is a key enzyme in the pyrimidine biosynthesis pathway involved in the mitochondrial electronic transport chain (mtETC).

Article Snippet: [ring‐ 14 C (U)] 4‐HB (50–60 mCi·mmol −1 ) was purchased from American Radiolabeled Chemicals (Saint Louis, Missouri, USA).

Techniques: Inhibition

Journal: Febs Letters

Article Title: 4‐nitrobenzoate inhibits 4‐hydroxybenzoate polyprenyltransferase in malaria parasites and enhances atovaquone efficacy

doi: 10.1002/1873-3468.70186

Figure Lengend Snippet: 4‐Hydroxybenzoate analogs effects on atovaquone and proguanil activity. (A) Structures of each of the 4‐HB analog tested. (B) Values of the IC 50 ± SD and R 2 values for each 4‐HB analog, as well as IC 50 values of AV and Proguanil in routine medium or medium supplemented with each compound, as indicated. Results represent the mean of three independent experiments. Green highlights 4‐NB. (C and D) Panels C and D display the results shown in Table B for AV and Proguanil, respectively. Results were analyzed using one‐way analysis of variance (ANOVA) with Dunnett's multiple comparison test compared to the control. * P < 0.05; ** P < 0.01; *** P < 0.001. Error bars indicate standard deviation (SD). The gray horizontal bar indicates FIC = 0.5, which corresponds to the threshold below which the effect is considered potentiation. Abbreviations: 4‐HB (4‐hydroxybenzoic acid), 4‐NB (4‐nitrobenzoic acid), 4‐ClB (4‐chlorobenzoic acid), 4‐BrB (4‐bromobenzoic acid), pABA (para‐aminobenzoic acid), PAS (para‐aminosalicylic acid), ns (statistically not significant), FIC (Fractional Inhibitory Concentration Index, which corresponds to the IC 50 value of atovaquone in medium supplemented with each 4‐HB analog divided by the IC 50 value of atovaquone alone).

Article Snippet: [ring‐ 14 C (U)] 4‐HB (50–60 mCi·mmol −1 ) was purchased from American Radiolabeled Chemicals (Saint Louis, Missouri, USA).

Techniques: Activity Assay, Comparison, Control, Standard Deviation, Concentration Assay

Journal: Febs Letters

Article Title: 4‐nitrobenzoate inhibits 4‐hydroxybenzoate polyprenyltransferase in malaria parasites and enhances atovaquone efficacy

doi: 10.1002/1873-3468.70186

Figure Lengend Snippet: PfCOQ2 complementation and enzymatic activity. (A) The figure shows the growth of yeast strains in SD + glucose/SD + glycerol media containing a concentration of 1 mM of different drugs, as indicated. This experiment was performed three times with similar results. (B) The figure shows the analysis of 4‐HPT enzymatic activity in PfCOQ2‐complemented yeasts in the presence of 0.5 mM of different drugs. The compounds added to the enzymatic reaction are indicated. Rf of 4‐HB and 3‐Farnesyl 4‐HB are also indicated. This experiment was performed three times with similar results. Abbreviations: 4‐NB (4‐nitrobenzoic acid), 4‐HB (4‐hydroxybenzoic acid), SD‐URA (synthetic defined medium without uracil), p416‐GPD vector (yeast expression plasmid carrying the constitutive GPD promoter, from the S. cerevisiae glyceraldehyde‐3‐phosphate dehydrogenase gene), COQ2 (4‐hydroxybenzoate polyprenyltransferase), Sc COQ2 ( Saccharomyces cerevisiae COQ2 gene), Pf COQ2 ( P. falciparum COQ2 gene), COQ2Δ‐0 (yeast strain with complete deletion of the COQ2 gene), FPP (farnesyl pyrophosphate), KO (knockout).

Article Snippet: [ring‐ 14 C (U)] 4‐HB (50–60 mCi·mmol −1 ) was purchased from American Radiolabeled Chemicals (Saint Louis, Missouri, USA).

Techniques: Activity Assay, Concentration Assay, Plasmid Preparation, Expressing, Knock-Out

Journal:

Article Title: Lysine 419 Targets Human Glucocorticoid Receptor for Proteasomal Degradation *

doi: 10.1016/j.steroids.2010.06.015

Figure Lengend Snippet: Glucocorticoid induction of reporter activity is enhanced by pretreatment with proteasome inhibitors in human GR transfected cells. COS-1 cells were transiently transfected with the pCMV-hGR human glucocorticoid receptor expression vector and the glucocorticoid responsive reporter GRE2-CAT. Cells were treated with vehicle control (Con) or 1 uM MG-132, β-lactone (Blact), or epoxomicin (Epox) for 1 hour and then treated with 100 nM dexamethasone (Dex) for 16 hours. Lysates were prepared for chloramphenicol acetyl transferase assay (CAT). Data are presented as CAT activity per ug of protein and represent the mean of three independent experiments. (* p<0.05 vs. CPM/ug Protein).

Article Snippet: [ 14 C] chloramphenicol (40–60 mCi/mmol) was obtained from NEN Life Science Products.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Chloramphenicol Acetyltransferase Assay

Journal:

Article Title: Lysine 419 Targets Human Glucocorticoid Receptor for Proteasomal Degradation *

doi: 10.1016/j.steroids.2010.06.015

Figure Lengend Snippet: Proteasome inhibition enhances endogenous human glucocorticoid receptor activity in HeLa cells treated with dexamethasone. HeLa cells were transiently transfected the glucocorticoid responsive reporter GRE2-CAT. Cells were treated with vehicle control (Con) or 1 uM MG-132 or epoxomicin (Epox) for 1 hour and then treated with 100 nM dexamethasone(Dex) for 16 hours. Lysates were prepared for chloramphenicol acetyl transferase assay (CAT). Equal ug of extract were used in each reaction. Data are presented as CAT activity per ug of protein and represent the mean of three independent experiments. (* p<0.05 vs. CAT activity/ug Protein)

Article Snippet: [ 14 C] chloramphenicol (40–60 mCi/mmol) was obtained from NEN Life Science Products.

Techniques: Inhibition, Activity Assay, Transfection, Control, Chloramphenicol Acetyltransferase Assay