Pierre Perio

Synthesis and antiplasmodial activity of new indolone N-oxide derivatives.

A series of 66 new indolone-N-oxide derivatives was synthesized with three different methods. Compounds were evaluated for in vitro activity against CQ-sensitive (3D7), CQ-resistant (FcB1), and CQ and pyrimethamine cross-resistant (K1) strains of Plasmodium falciparum (P.f.), as well as for cytotoxic concentration (CC(50)) on MCF7 and KB human tumor cell lines. Compound 26 (5-methoxy-indolone-N-oxide analogue) had the most potent antiplasmodial activity in vitro (<3 nM on FcB1 and = 1.7 nM on 3D7) with a very satisfactory selectivity index (CC(50) MCF7/IC(50) FcB1: 14623; CC(50) KB/IC(50) 3D7: 198823). In in vivo experiments, compound 1 (dioxymethylene derivatives of the indolone-N-oxide) showed the best antiplasmodial activity against Plasmodium berghei, 62% inhibition of the parasitaemia at 30 mg/kg/day.

Interaction between antimalarial 2-aryl-3H-indol-3-one derivatives and human serum albumin.

Binding of drugs to plasma proteins, such as albumin, is a major factor which determines their pharmacokinetics and pharmacological effects. Therefore, the interactions between human serum albumin (HSA) and four antimalarial compounds selected in the 2-aryl-3H-indol-3-one series have been investigated using UV-visible, fluorescence and circular dichroism (CD) spectroscopies. Compounds produced a static quenching of the intrinsic fluorescence of HSA. The thermodynamic parameters have shown that the binding reaction is endothermic for three compounds while exothermic for the 2-phenyl-3H-indol-3-one, 3. The interaction is entropically driven with predominant hydrophobic forces with binding affinities of the order of 10(4) M(-1). The highest binding constant is observed for 3 (Kλ=280nm = 4.53 × 10(4) M(-1)) which is also the less active compound against Plasmodium falciparum. Synchronous fluorescence gave qualitative information on the conformational changes of HSA while quantitative data were obtained with CD. Displacement experiments with site markers indicated that drugs bind to HSA at site I (subdomain IIA). In addition, the apparent binding constant and the binding site number were calculated in the presence of different ions.

Insights into the redox cycle of human quinone reductase 2

Abstract NRH:quinone oxidoreductase 2 (QR2) is a cytosolic enzyme that catalyzes the reduction of quinones, such as menadione and co-enzymes Q. With the aim of understanding better the mechanisms of action of QR2, we approached this enzyme catalysis via electron paramagnetic resonance (EPR) measurements of the by-products of the QR2 redox cycle. The variation in the production of oxidative species such as H2O2, and subsequent hydroxyl radical generation, was measured during the course of QR2 activity under aerobic conditions and using pure human enzyme. The effects on the activity of the following were compared: (i) synthetic (N-benzyldihydronicotinamide, BNAH) or natural (nicotinamide riboside, NRH) co-substrates; (ii) synthetic (menadione) or natural (co-enzyme Q0, Q2) substrates; (iii) QR2 modulators and inhibitors (melatonin, resveratrol and S29434); (iv) a pro-drug activated via a redox cycle [CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide]. The results were also compared with those obtained with human QR1. The production of hydroxyl radicals is: (i) observed whatever the substrate/co-substrate used; ii) quenched by adding catalase; (iii) not observed with the specific QR2 inhibitor S29434; (iv) observed with the pro-drug CB1954. While QR2 produced free radicals with this pro-drug, QR1 gave no EPR signal showing the strong reducing capacity of QR2. In conclusion, EPR analysis of QR2 enzyme activity through free radical production enables modulators and effective inhibitors to be distinguished.

Electrochemical behavior of indolone-N-oxides: Relationship to structure and antiplasmodial activity

Indolone-N-oxides exert high parasiticidal activity at the nanomolar level in vitro against Plasmodium falciparum, the parasite responsible for malaria. The bioreductive character of these molecules was investigated using cyclic voltammetry and EPR spectroelectrochemistry to examine the relationship between electrochemical behavior and antimalarial activity and to understand their mechanisms of action. For all the compounds (37 compounds) studied, the voltammograms recorded in acetonitrile showed a well-defined and reversible redox couple followed by a second complicated electron transfer. The first reduction (-0.88V<E(1/2)<-0.50V vs. SCE) was attributed to the reduction of the N-oxide function to form a radical nitroxide anion. The second reduction (-1.65V<E(1/2)<-1.14V vs. SCE) was assigned to the reduction of the ketone function. By coupling electrochemistry with EPR spectroscopy, the EPR spectra confirmed the formation of the nitroxide anion radical. Moreover, the experiments demonstrated that a slow protonation occurs at the carbon of the nitrone function and not at the NO function. A relationship between electrochemical behavior and indolone-N-oxide structure can be established for compounds with R(1)=-OCH(3), R(2)=H, and electron-withdrawing substituents on the phenyl group at R(3). The results help in the design of new molecules with more potent in vivo antimalarial activity.

In cellulo monitoring of quinone reductase activity and reactive oxygen species production during the redox cycling of 1,2 and 1,4 quinones.

Quinones are highly reactive molecules that readily undergo either one- or two-electron reduction. One-electron reduction of quinones or their derivatives by enzymes such as cytochrome P450 reductase or other flavoproteins generates unstable semiquinones, which undergo redox cycling in the presence of molecular oxygen leading to the formation of highly reactive oxygen species. Quinone reductases 1 and 2 (QR1 and QR2) catalyze the two-electron reduction of quinones to form hydroquinones, which can be removed from the cell by conjugation of the hydroxyl with glucuronide or sulfate thus avoiding its autoxidation and the formation of free radicals and highly reactive oxygen species. This characteristic confers a detoxifying enzyme role to QR1 and QR2, even if this character is strongly linked to the excretion capacity of the cell. Using EPR spectroscopy and confocal microscopy we demonstrated that the amount of reactive oxygen species (ROS) produced by Chinese hamster ovary (CHO) cells overexpressing QR1 or QR2 compared to naive CHO cells was determined by the quinone structural type. Indeed, whereas the amount of ROS produced in the cell was strongly decreased with para-quinones such as menadione in the presence of quinone reductase 1 or 2, a strong increase in ROS was recorded with ortho-quinones such as adrenochrome, aminochrome, dopachrome, or 3,5-di-tert-butyl-o-benzoquinone in cells overexpressing QR, especially QR2. These differences could originate from the excretion process, which is different for para- and ortho-quinones. These results are of particular interest in the case of dopamine considering the association of QR2 with various neurological disorders such as Parkinson disease.

Dereplication of natural products from complex extracts by regression analysis and molecular networking: case study of redox-active compounds from Viola alba subsp. dehnhardtii

IntroductionIn natural product research, bioassay-guided fractionation was previously widely employed but is now judged to be inadequate in terms of time and cost, particularly if only known compounds are ultimately isolated. The development of metabolomics, along with improvements in analytical tools, allows comprehensive metabolite profiling. This enables dereplication to target unknown active compounds early in the purification workflow.ObjectivesStarting from an ethanolic extract of violet leaves, this study aims to predict redox active compounds within a complex matrix through an untargeted metabolomics approach and correlation analysis.MethodsRapid fractionation of crude extracts was carried out followed by multivariate data analysis (MVA) of liquid chromatography–high resolution mass spectrometry (LC–HRMS) profiles. In parallel, redox active properties were evaluated by the capacity of the molecules to reduce 2,2-diphenyl-1-picrylhydrazyl (DPPH·) and superoxide (O2·−) radicals using UV–Vis and electron spin resonance spectroscopies (ESR), respectively. A spectral similarity network (molecular networking) was used to highlight clusters involved in the observed redox activities.ResultsDereplication on Viola alba subsp. dehnhardtii highlighted a reproducible pool of redox active molecules. Polyphenols, particularly O-glycosylated coumarins and C-glycosylated flavonoids, were identified and de novo dereplicated through molecular networking. Confirmatory analyses were undertaken by thin layer chromatography (TLC)–DPPH–MS assays and nuclear magnetic resonance (NMR) spectra of the most active compounds.ConclusionOur dereplication strategy allowed the screening of leaf extracts to highlight new biologically active metabolites in few steps with a limited amount of crude material and reduced time-consuming manipulations. This approach could be applied to any kind of natural extract for the study of various biological activities.

Pro-oxidant properties of indolone-N-oxides in relation to their antimalarial properties

Indolone-N-oxides (INODs) are bioreducible and possess remarkable anti-malarial activities in the low nanomolar range in vitro against different Plasmodium falciparum (P. falciparum) strains and in vivo. INODs have an original mechanism of action: they damage the host cell membrane without affecting non-parasitized erythrocytes. These molecules produce a redox signal which activates SYK tyrosine kinases and induces a hyperphosphorylation of AE1 (band 3, erythrocyte membrane protein). The present work aimed to understand the early stages of the biochemical interactions of these compounds with some erythrocyte components from which the redox signal could originate. The interactions were studied in a biomimetic model and compared with those of chloroquine and artemisinin. The results showed that INODs i) do not enter the coordination sphere of the metal in the heme iron complex as does chloroquine; ii) do not generate iron-dependent radicals as does artemisinin; iii) generate stable free radical adducts after reduction at one electron; iv) cannot trap free radicals after reduction. These results confirm that the bioactivity of INODs does not lie in their spin-trapping properties but rather in their pro-oxidant character. This property may be the initiator of the redox signal which activates SYK tyrosine kinases.

EPR spectroelectrochemical investigation of guanine radical formation and environment effects.

Guanine radical detection was carried out by a new convenient and efficient method coupling electron paramagnetic resonance spectroscopy and indirect electrooxidation of guanine in different biological environments, from the free nucleotide to several types of DNA substrates. Compared to the widely used photoirradiation method, this method appeared more selective in the choice of the electrochemical mediator. Carried out in presence of a ruthenium mediator and PBN as spin trap, this method revealed two types of EPR spectra depending of the environment of the guanine radical. Both EPR spectra show the trapping of the neutral guanine radical G(-H)(•) obtained after fast deprotonation of the radical cation G(•+). However, they differ by the atom where the trapped radical is centered. This difference highlights the structural dependency of the environment on the nature of the radical formed. This work gave the evidence of an innovative method to detect in situ the guanine radical.

Role of Quinone Reductase 2 in the Antimalarial Properties of Indolone-Type Derivatives

Indolone-N-oxides have antiplasmodial properties against Plasmodium falciparum at the erythrocytic stage, with IC50 values in the nanomolar range. The mechanism of action of indolone derivatives involves the production of free radicals, which follows their bioreduction by an unknown mechanism. In this study, we hypothesized that human quinone reductase 2 (hQR2), known to act as a flavin redox switch upon binding to the broadly used antimalarial chloroquine, could be involved in the activity of the redox-active indolone derivatives. Therefore, we investigated the role of hQR2 in the reduction of indolone derivatives. We analyzed the interaction between hQR2 and several indolone-type derivatives by examining enzymatic kinetics, the substrate/protein complex structure with X-ray diffraction analysis, and the production of free radicals with electron paramagnetic resonance. The reduction of each compound in cells overexpressing hQR2 was compared to its reduction in naïve cells. This process could be inhibited by the specific hQR2 inhibitor, S29434. These results confirmed that the anti-malarial activity of indolone-type derivatives was linked to their ability to serve as hQR2 substrates and not as hQR2 inhibitors as reported for chloroquine, leading to the possibility that substrate of hQR2 could be considered as a new avenue for the design of new antimalarial compounds.

Powerful Antioxidant and Pro-Oxidant Properties of Cassia roxburghii DC. Leaves Cultivated in Egypt in Relation to Their Anti-Infectious Activities

Leaf extracts of Cassia roxburghii DC., prepared in petroleum ether, chloroform, ethyl acetate, butanol, and methanol/water (70:30, v/v), were evaluated as antioxidant, pro-oxidant, anti-infectious, and cytotoxic agents. The major metabolite of each extract was identified by chromatographic and spectroscopic means. The redox properties were assessed with a battery of assays, which revealed that the ethyl acetate extract demonstrated an interesting scavenging activity of DPPH and superoxide radicals and an ascorbic acid-like pro-oxidant activity. All the tested extracts showed moderate antiplasmodial activity against a chloroquine-resistant strain of Plasmodium falciparum, by possible disruption of parasite fine redox balance. Cytotoxicity was evaluated against a human breast cancer cell line. The antimicrobial activities of the extracts were estimated against representative bacterial strains (Staphylococcus aureus, Enterococcus hirae, Pseudomonas aeruginosa, Escherichia coli) and fungal species (Candida albicans, Aspergillus niger). The ethylacetate extract possessed the highest redox properties and exhibited the highest antiplasmodial activity; there was no correlation between antibacterial activity and the redox properties of the extracts.