Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells.

Redox-sensitive green fluorescent protein 2 (roGFP2) is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are shown to correlate with the ratiometrically measured degree of oxidation of corresponding roGFP2 fusion proteins. Furthermore, stopped-flow kinetic measurements of the oxidative half-reaction of PfAOP support the interpretation that changes in the roGFP2 signal can be used to map hyperoxidation-based inactivation of the attached peroxidase. Potential future applications of our system include the improvement of redox sensors, the estimation of absolute intracellular peroxide concentrations and the in vivo assessment of protein structure-function relationships that cannot easily be addressed with recombinant enzymes, for example, the effect of post-translational protein modifications on enzyme catalysis.

Knockout of the peroxiredoxin 5 homologue PFAOP does not affect the artemisinin susceptibility of Plasmodium falciparum.

Artemisinins are the current mainstay of malaria chemotherapy. Their exact mode of action is an ongoing matter of debate, and several factors have recently been reported to affect an early stage of artemisinin resistance of the most important human malaria parasite Plasmodium falciparum. Here, we identified a locus on chromosome 7 that affects the artemisinin susceptibility of P. falciparum in a quantitative trait locus analysis of a genetic cross between strains 7G8 and GB4. This locus includes the peroxiredoxin gene PFAOP. However, steady-state kinetic data with recombinant PfAOP do not support a direct interaction between this peroxidase and the endoperoxide artemisinin. Furthermore, neither the overexpression nor the deletion of the encoding gene affected the IC50 values for artemisinin or the oxidants diamide and tert-butyl hydroperoxide. Thus, PfAOP is dispensable for blood stage parasite survival, and the correlation between the artemisinin susceptibility and chromosome 7 is probably based on another gene within the identified locus.

Growth inhibitory effects of standard pro- and antioxidants on the human malaria parasite Plasmodium falciparum.

The redox metabolism of the malaria parasite Plasmodium falciparum and its human host has been suggested to play a central role for parasite survival and clearance. A common approach to test hypotheses in redox research is to challenge or rescue cells with pro- and antioxidants. However, quantitative data on the susceptibility of infected erythrocytes towards standard redox agents is surprisingly scarce. Here we determined the IC50 values of P. falciparum strains 3D7 and Dd2 for a set of redox agents using a SYBR green-based growth assay. Parasite killing in this assay required extremely high concentrations of hydrogen peroxide with a millimolar IC50 value, whereas IC50 values for tert-butyl hydroperoxide and diamide were between 67 and 121 µM. Thus, in contrast to tert-butyl hydroperoxide and the disulfide-inducing agent diamide, the host-parasite unit appears to be very robust against challenges with hydrogen peroxide with implications for host defense mechanisms. N-acetylcysteine, ascorbate, and dithiothreitol also had antiproliferative instead of growth-promoting effects with IC50 values around 12, 3 and 0.4 mM, respectively. So-called antioxidants can therefore also inhibit parasite growth with implications for clinical trials and studies on 'oxidative stress'. Furthermore, the addition of reductants to parasite cultures resulted in the gelation of albumin, the formation of methemoglobin and hemolysis. These effects can alter the fluorescence in SYBR green assays and have to be taken into account for the determination of IC50 values. In summary, standard oxidants and reductants both inhibit the growth of P. falciparum with IC50 values differing by three orders of magnitude.

The cytosolic glyoxalases of Plasmodium falciparum are dispensable during asexual blood-stage development.

The enzymes glyoxalase 1 and 2 (Glo1 and Glo2) are found in most eukaryotes and catalyze the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Four glyoxalases are encoded in the genome of the malaria parasite Plasmodium falciparum, the cytosolic enzymes PfGlo1 and PfcGlo2, the apicoplast enzyme PftGlo2, and an inactive Glo1-like protein that also carries an apicoplast-targeting sequence. Inhibition or knockout of the Plasmodium glyoxalases was hypothesized to lead to an accumulation of 2-oxoaldehydes and advanced glycation end-products (AGE) in the host-parasite unit and to result in parasite death. Here, we generated clonal P. falciparum strain 3D7 knockout lines for PFGLO1 and PFcGLO2 using the CRISPR-Cas9 system. Although 3D7Δglo1 knockout clones had an increased susceptibility to external glyoxal, all 3D7Δglo1 and 3D7Δcglo2 knockout lines were viable and showed no significant growth phenotype under standard growth conditions. Furthermore, the lack of PfcGlo2, but not PfGlo1, increased gametocyte commitment in the knockout lines. In summary, PfGlo1 and PfcGlo2 are dispensable during asexual blood-stage development while the loss of PfcGlo2 may induce the formation of transmissible gametocytes. These combined data show that PfGlo1 and PfcGlo2 are most likely not suited as targets for selective drug development.

Plasmodium falciparum antioxidant protein reveals a novel mechanism for balancing turnover and inactivation of peroxiredoxins.

Life under aerobic conditions has shaped peroxiredoxins (Prx) as ubiquitous thiol-dependent hydroperoxidases and redox sensors. Structural features that balance the catalytically active or inactive redox states of Prx, and, therefore, their hydroperoxidase or sensor function, have so far been analyzed predominantly for Prx1-type enzymes. Here we identify and characterize two modulatory residues of the Prx5-type model enzyme PfAOP from the malaria parasite Plasmodium falciparum. Gain- and loss-of-function mutants reveal a correlation between the enzyme parameters and the inactivation susceptibility of PfAOP with the size of residue 109 and the presence or absence of a catalytically relevant but nonessential cysteine residue. Based on our kinetic data and the crystal structure of PfAOP(L109M), we suggest a novel mechanism for balancing the hydroperoxidase activity and inactivation susceptibility of Prx5-type enzymes. Our study provides unexpected insights into Prx structure-function relationships and contributes to our understanding of what makes Prx good enzymes or redox sensors.

Bioorthogonal prodrug activation driven by a strain-promoted 1,3-dipolar cycloaddition.

Due to the formation of hydrolysis-susceptible adducts, the 1,3-dipolar cycloaddition between an azide and strained trans-cyclooctene (TCO) has been disregarded in the field of bioorthogonal chemistry. We report a method which uses the instability of the adducts to our advantage in a prodrug activation strategy. The reaction of trans-cyclooctenol (TCO-OH) with a model prodrug resulted in a rapid 1,3-dipolar cycloaddition with second-order rates of 0.017 M-1 s-1 and 0.027 M-1 s-1 for the equatorial and axial isomers, respectively, resulting in release of the active compound. 1H NMR studies showed that activation proceeded via a triazoline and imine, both of which are rapidly hydrolyzed to release the model drug. Cytotoxicity of a doxorubicin prodrug was restored in vitro upon activation with TCO-OH, while with cis-cyclooctenol (CCO-OH) no activation was observed. The data also demonstrates the potential of this reaction in organic synthesis as a mild orthogonal protecting group strategy for amino and hydroxyl groups.

Systematic re-evaluation of the bis(2-hydroxyethyl)disulfide (HEDS) assay reveals an alternative mechanism and activity of glutaredoxins.

The reduction of bis(2-hydroxyethyl)disulfide (HEDS) by reduced glutathione (GSH) is the most commonly used assay to analyze the presence and properties of enzymatically active glutaredoxins (Grx), a family of central redox proteins in eukaryotes and glutathione-utilizing prokaryotes. Enzymatically active Grx usually prefer glutathionylated disulfide substrates. These are converted via a ping-pong mechanism. Sequential kinetic patterns for the HEDS assay have therefore been puzzling since 1991. Here we established a novel assay and used the model enzyme ScGrx7 from yeast and PfGrx from Plasmodium falciparum to test several possible causes for the sequential kinetics such as pre-enzymatic GSH depletion, simultaneous binding of a glutathionylated substrate and GSH, as well as substrate or product inhibition. Furthermore, we analyzed the non-enzymatic reaction between HEDS and GSH by HPLC and mass spectrometry suggesting that such a reaction is too slow to explain high Grx activities in the assay. The most plausible interpretation of our results is a direct Grx-catalyzed reduction of HEDS. Physiological implications of this alternative mechanism and of the Grx-catalyzed reduction of non-glutathione disulfide substrates are discussed.

Synthesis and Antifungal Evaluation of Novel N-Alkyl Tetra- and Perhydroquinoline Derivatives.

A series of novel N-alkyl tetra- and perhydroquinoline derivatives and their hydrochlorides were prepared from tetrahydro- or trans-perhydroquinoline by direct alkylation with alkyl halides and subsequent precipitation with HCl gas. The antimicrobial activity of the resulting amines was evaluated in an agar diffusion assay. The minimal inhibitory concentrations (MIC) of the active compounds were determined by the microdilution method. In contrast to the tetrahydroquinolines, the perhydro analogues showed significant antifungal activity. In an assay for the detection of target enzymes in ergosterol biosynthesis, N-undecylperhydroquinoline was identified as an inhibitor of Δ8,7-isomerase.

Synthesis and biological evaluation of novel N-alkyl tetra- and decahydroisoquinolines: novel antifungals that target ergosterol biosynthesis.

A series of N-alkyl trans-decahydroisoquinoline, 1,2,3,4-tetrahydroisoquinoline, and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivatives were synthesized starting from the respective secondary amines by N-alkylation with alkyl bromides. The compounds with C11-alkyl chains showed antifungal potency comparable to clotrimazole, and inhibit enzymes of the ergosterol biosynthesis (Δ14-reductase and Δ8,7-isomerase), depending on the heterocyclic scaffold and the investigated species.

Synthesis and Biological Evaluation of Novel Alkyl-Imidazolyl Carbinols and their Esters: Potent Antimycotics.

A novel series of imidazol-5-yl carbinols and their 4-chlorobenzoyl esters has been synthesized by the Grignard reaction and subsequent esterification. These compounds were screened for their antimicrobial activities in an agar diffusion assay. The compounds with C10 to C12-alkyl side chains displayed significant antimycotic activity.

A convenient cellular assay for the identification of the molecular target of ergosterol biosynthesis inhibitors and quantification of their effects on total ergosterol biosynthesis.

Increasing resistance of clinically relevant fungi is causing major problems in anti-mycotic therapy. Particularly for immunosuppressed patients fungal infections are of concern and increasing resistance against clinically used antimycotic drugs is hampering successful treatment. In the search for new antifungals ergosterol biosynthesis still is the most prominent target. However, several pitfalls in the bioactivity testing of such substances remain. Two of the major drawbacks certainly are the membrane association of most enzymes participating in ergosterol biosynthesis, and the difficulty to selectively associate growth inhibitory effects with the target pathway (ergosterol biosynthesis). Here we describe a GC-MS based cellular assay for target identification and selective potency determination of test components. In the qualitative part of the assay GC-MS analysis of cell lysates allows target identification by analysis of the changes in the sterol pattern. The quantitative part of the assay makes use of 13C-acetate feeding combined with GC-MS analysis allowing the selective quantification of a compound's effect on total ergosterol biosynthesis. The described cellular assay was analytically and biologically validated and used to characterize the novel ergosterol biosynthesis inhibitor JK-250.