Jean Bernadou

DNA And RNA Cleavage by Metal Complexes

Publisher Summary This chapter discusses DNA and RNA cleavage by metal complexes. DNA and RNA cleavage, a very active field of research, has been developed in two main and complementary directions within the past decade: oxidative cleavage and hydrolysis. In general, the difference between the two different approaches are (1) the preparation of new chemical tools to study genomic DNA. The recognition sites of most of the restriction enzymes are often limited to palindromic sequences, and it is useful to have artificial nucleases able to cleave DNA at any desired sequence, and (2), the synthesis of compounds that cleave nucleic acids should help in the design of potential therapeutic agents for the treatment of cancer and viral diseases. The challenging development of new, efficient DNA and RNA cleavage agents can require a strong cooperation between chemists, biochemists, and molecular biologists.

Mn(III) Pyrophosphate as an Efficient Tool for Studying the Mode of Action of Isoniazid on the InhA Protein of Mycobacterium tuberculosis

ABSTRACT The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III) pyrophosphate. In the presence of nicotinamide coenzymes (NAD+, NADH, nicotinamide mononucleotide [NMN+]) and nicotinic acid adenine dinucleotide (DNAD+), INH oxidation produced the formation of INH-coenzyme adducts in addition to known biologically inactive products (isonicotinic acid, isonicotinamide, and isonicotinaldehyde). A pool of INH-NAD(H) adducts preformed in solution allowed the rapid and strong inhibition of in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids: the inhibition was 90 or 60% when the adducts were formed in the presence of NAD+ or NADH, respectively. Under similar conditions, no inhibitory activity of INH-NMN(H) and INH-DNAD(H) adducts was detected. When an isolated pool of 100 nM INH-NAD(H) adducts was first incubated with InhA, the enzyme activity was inhibited by 80%; when present in excess, both NADH and decenoyl-coenzyme A are able to prevent this phenomenon. InhA inhibition by several types of INH-coenzyme adducts coexisting in solution is discussed in relation with the structure of the coenzyme, the stereochemistry of the adducts, and their existence as both open and cyclic forms. Thus, manganese(III) pyrophosphate appears to be an efficient and convenient alternative oxidant to mimic the activity of the Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations of reactive INH species in order to promote the design of new inhibitors of InhA as potential antituberculous drugs.

Active Iron-Oxo and Iron-Peroxo Species in Cytochromes P450 and Peroxidases; Oxo-Hydroxo Tautomerism with Water-Soluble Metalloporphyrins

Heme-containing monooxygenases are able to catalyze two different classes of oxidation reactions. The first class includes oxygenation reactions (hydroxylation, epoxidation, N- or S-oxide formation, etc.) which are mediated by an electrophilic oxidative species. The second class is represented by the oxidative deformylation of aldhehydes and involves a nucleophilic oxidant as active intermediate. The reductive activation of molecular oxygen by cytochromes P450 generates a nucleophilic iron(III)-peroxo species which produces by protonation an electrophilic high-valent iron-oxo [formally an iron(V)oxo] responsible for electrophilic oxygen atom transfers. The nucleophilic properties of the iron(III)-peroxo intermediate in cytochrome P450 are due to the porphyrin ring acting as electron reservoir and also to the negative charge accumulated on the proximal cysteine during the initial reduction step of the catalytic cycle. The nature of the high-valent iron-oxo species generated in the catalytic cycle of heme-peroxidases will be also discussed. Among the different methods for studying the oxygenation reactions mediated by high-valent metal-oxo porphyrin complexes, the recent discovery of the “oxo-hydroxo tautomerism” provides a useful tool to investigate the mechanism of O-atom transfer reactions in aqueous media.

A Fast and Efficient Metal‐Mediated Oxidation of Isoniazid and Identification of Isoniazid–NAD(H) Adducts

It is currently believed that isoniazid (INH) is oxidised inside Mycobacterium tuberculosis to generate, by covalent attachment to the nicotinamide ring of NAD(H) (β‐nicotinamide adenine dinucleotide), a strong inhibitor of InhA, an enzyme essential for mycolic acid biosynthesis. This work was carried out to characterise the InhA inhibitors (named INH–NAD(H) adducts) which are generated, in the presence of the nicotinamide coenzyme NAD+, by oxidation of INH with manganese(III) pyrophosphate, a nonenzymatic and efficient oxidant used to mimic INH activation by the catalase–peroxidase KatG inside M. tuberculosis. The oxidation process is almost complete in less than 15 minutes (in comparison to the slow activation obtained in the KatG‐dependent process (2.5 hours) or in the nonenzymatic O2/MnII‐dependent activation (5 hours)). The alkylation of NAD+ by the postulated isonicotinoyl radical generates, in solution, a family of INH–NAD(H) adducts. Analyses with liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI‐MS) and experiments performed with 18O‐ and 2H‐labelled substrates allowed us to propose two open and four hemiamidal cyclised dihydropyridine structures as the main forms present in solution; these result from the combination of the isonicotinoyl radical and the nicotinamide part of NAD+. A small amount of a secondary oxidation product was also detected. Structural data on the forms present in solution should help in the design of inhibitors of enzymes involved in the biosynthesis of mycolic acids to act as potential antituberculosis drugs.

In Vitro Inhibition of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA by Isoniazid

ABSTRACT The first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present work shows that the NADPH-dependent β-ketoacyl reduction activity of MabA is efficiently inhibited by isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the formation of a covalent adduct between MnIII-activated isoniazid and the MabA cofactor. Liquid chromatography-mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand complex strongly suggested that the molecules active against MabA activity are the oxidized derivative and a major cyclic form. Spectrofluorimetry showed that the adduct binds to the MabA active site. Modeling of the MabA-adduct complex predicted an interaction between the isonicotinoyl moiety of the inhibitor and Tyr185. This hypothesis was supported by the fact that a higher 50% inhibitory concentration of the adduct was measured for MabA Y185L than for the wild-type enzyme, while both proteins presented similar affinities for NADP+. The crystal structure of MabA Y185L that was solved showed that the substitution of Tyr185 induced no significant conformational change. The description of the first inhibitor of the β-ketoacyl reduction step of fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrug-resistant tubercle bacilli.

New approach to metabolism of 5'-deoxy-5-fluorouridine in humans with fluorine-19 NMR

SummaryThe metabolism of 5′-deoxy-5-fluorouridine (5′dFUrd), an antitumor fluoropyrimidine, has been investigated in human biofluids (blood, plasma, urine) using a new method: fluorine-19 NMR spectrometry. This method allows direct study of the biological sample and simultaneous identification of all the fluorinated metabolites. In the blood of a patient treated with 5′dFUrd during a 6-h continuous perfusion, we observed unmetabolized 5′dFUrd, 5-fluorouracil, 5,6-dihydrofluorouracil, and another metabolite which has not previously been reported α-fluoro-β-alanine. The two major metabolites in urine are unmetabolized 5′dFUrd and α-fluoroβ-alanine.

Metal-oxo species in P450 enzymes and biomimetic models. Oxo-hydroxo tautomerism with water-soluble metalloporphyrins

Oxygenation reactions (hydroxylation, epoxidation, N- or S-oxide formation, etc.) catalyzed by cytochrome P450 enzymes and related biomimetic models involve an electrophilic oxidative species as the active species, namely a high-valent metal-oxo intermediate. Among the different methods to study the oxygenation reactions mediated by high-valent metal-oxo porphyrin complexes, the recent discovery of oxo-hydroxo tautomerism provides a useful tool to investigate the mechanism of O-atom transfer reactions in aqueous media.

The Ligand 1,10-Phenanthroline-2,9-dicarbaldehyde Dioxime can Act Both as a Tridentate and as a Tetradentate Ligand − Synthesis, Characterization and Crystal Structures of its Transition Metal Complexes

The polydentate ligand L has been prepared by treating 1,10-phenanthroline-2,9-dicarbaldehyde with hydroxylamine. This ligand and its complexes with ZnII, CuII, CdII, CoII and NiII were designed as potential agents for nucleic acid hydrolysis and have been characterized by elemental analysis, NMR spectroscopy, ES-MS and X-ray diffraction analysis. The ligand acts as a tridentate ligand in most cases, to form monomeric trigonal-bipyramidal, square-pyramidal or square-bipyramidal structures. In the case of metallation with cadmium nitrate it acts as a tetradentate ligand to give a pentagonal-bipyramidal structure. Dissociation of the oxime group to an oximate, which acts as an anionic bridging ligand, was observed in the case of metallation with zinc acetate thus giving rise to dimers and/or polymers.

DNA strand breaks photosensitized by benoxaprofen and other non steroidal antiinflammatory agents.

Benoxaprofen, a non steroidal antiinflammatory drug is known to be highly phototoxic. Upon irradiation at 300 nm, benoxaprofen is shown to enhance the cleavage of phi X 174 DNA in buffered aqueous solution (pH 7.4). A linear relationship between the number of single strand breaks and the irradiation time is found. In deaerated solutions, these breaks are three times greater in the presence than in the absence of benoxaprofen. In both cases the rate of cleavage decreases in the presence of air. The rate of DNA damage increases with the drug per base pair ratio up to approximatively 0.2 and then decreases at higher ratios. Other NSAIDs, naproxen, ketoprofen, diflunisal, sulindac and indomethacin have been tested as photocleavers of DNA by using the same experimental conditions. A comparison of the efficiency of cleavage of all these drugs (including BNP) was obtained at drug concentrations such that the light absorbance was the same. Benoxaprofen, naproxen, ketoprofen and diflunisal induce single strand breaks. Sulindac and indomethacin do not cause breaks, and they can in some conditions even act as screening agents. The most efficient of the series are naproxen and ketoprofen. In the presence of oxygen, at the same concentrations as above, the efficiency of benoxaprofen, ketoprofen and diflunisal is decreased while that of naproxen is increased. This suggests that all these compounds do not interact with DNA by the same mechanism. In the case of BNP, the mechanism of photoinduced DNA cleavage is discussed in detail. It is shown that the photoactive agent is the decarboxylated derivative of benoxaprofen, as the photodecarboxylation of benoxaprofen is much faster than the photocleavage of DNA.

Development of isoniazid-NAD truncated adducts embedding a lipophilic fragment as potential bi-substrate InhA inhibitors and antimycobacterial agents.

Isoniazid-NAD truncated adducts embedding a lipophilic fragment were designed, synthesized and evaluated as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (InhA) of Mycobacterium tuberculosis and as antimycobacterial agents. These compounds, planned as bi-substrate inhibitors and inspired from the active metabolite of isoniazid, combine both the nicotinamide moiety of the cofactor NAD and a lipophilic hydrocarbon chain mimic of the InhA substrate. The lipophilic fragment was introduced using either Suzuki-Miyaura cross-coupling or a classical nucleophilic substitution reaction. Several compounds developed in this work were indeed able to inhibit the InhA activity and showed promising antimycobacterial activities. However a direct correlation between the expressed activity and the bi-substrate mode of action could not yet be unambiguously demonstrated.