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Five lipophilic analogues 1–5 of the active metabolite of the antitubercular drug isoniazid (INH), selected as inhibitors of Mycobacterium smegmatis and Mycobacterium tuberculosis growth, were evaluated for their activity against Corynebacterium glutamicum (lacking in InhA activity), Escherichia coli (to test mycobacteria selectivity), and Plasmodium falciparum (as possible parasite target). Compound 3 was the only one that did not inhibit C. glutamicum growth. The poor InhA inhibitors 1 and 2 were able to inhibit C. glutamicum and their anti(myco)bacterial mechanisms of action involve targets other than InhA. For the effective InhA inhibitors 4 and 5, also active against C. glutamicum and M. tuberculosis strains, more than one pathway should be envisaged to explain their actions. Pyridine-base ring analogues (1, 2, and 3) have no ability to inhibit the growth of E. coli even at a high concentration. Compound 3 thus exhibited a selective inhibitory action toward M. tuberculosis, while it was inactive on C. glutamicum and on E. coli growth. It presented an activity profile similar to that of INH suggesting InhA inhibition as one of the possible mechanisms of action. Finally, although a homologue of the reductase InhA exists in the FAS-II system of P. falciparum, 3 was unable to display antiplasmodial activity.

Porphyrins are used as photosensitizer (PS) in photodynamic therapy in cancer treatment. Nevertheless, the development of photochemotherapy in oncology remains limited, because of the low selectivity of PSs. In order to allow PS targeting toward tumor-associated antigens, for the first time a white-light activatable porphyrin, [5-(4-(5-carboxy-1-butoxy)-phenyl)-10,15,20-tris(4-N-methyl)-pyridiniumyl)-porphyrin] (TrMPyP) was covalently linked to Morniga G (MorG), a galactose-specific binding plant lectin, known to recognize with high-affinity tumor-associated T/Tn antigen in cell-free systems. Firstly, using fluorescein-labeled MorG, the sugar-dependent binding and uptake of lectin by Tn-positive (Jurkat lymphoid leukemia) cells was demonstrated. Secondly, the TrMPyP-MorG conjugate was molecularly characterized. Cytometric and confocal microscopic analysis demonstrated that PS covalent linking to MorG preserved sugar-dependent specific binding and uptake of lectin by Jurkat leukemia lymphocytes. Thirdly, the conjugate (with a 1:1 PS:lectin ratio) that was bound and quickly (5 min) taken-up, induced greater than 90% cytotoxicity upon irradiation at 10 nm concentration, whereas the free PS was absolutely nontoxic. On the contrary, normal lymphocytes strongly resisted to the conjugate-mediated phototoxicity. Thus, owing to their binding and endocytosis capacities, plant lectins represent promising molecules for targeting of tumor glycan alteration and to enhance the efficiency of specific delivery of PSs to tumor cells.

The front-line antituberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis enoyl ACP-reductase from Mycobacterium tuberculosis, via formation of covalent adducts with NAD (INH-NAD adducts). While ring tautomers were found the main species formed in solution, only the 4S chain INH-NAD tautomer was evidenced in the crystallized InhA:INH-NAD complex. In this study we attempted to explore the modes of interaction and energy binding of the different isomers placed in the active site of InhA with the help of various molecular modelling techniques. Ligand and enzyme models were generated with the help of the Vega ZZ program package. Resulting ligands were then docked into the InhA active site individually using computational automated docking package AUTODOCK 3.0.5. The more relevant docked conformations were then used to compute the interaction energy between the ligands and the InhA cavity. The AM1 Hamiltonian and the QM/MM ONIOM methodologies were used and the results compared. The various tautomers were found docked in almost the same place where INH-NAD was present as predicted by earlier X-ray crystallographic studies. However, some changes of ligand conformation and of the interactions ligand-protein were evidenced. The lower binding energy was observed for the 4S chain adduct that probably represents the effective active form of the INH-NAD adducts, as compared to the 4R epimer. The two 4S,7R and 4R,7S ring tautomers show intermediate and similar binding energies contrasting with their different experimental inhibitory potency on InhA. As a possible explanation based on calculated conformations, we formulated the hypothesis of an initial binding of the two ring tautomers to InhA followed by opening of only the ring hemiamidal 4S,7R tautomer (possibly catalyzed by Tyr158 phenolate basic group) to give the 4S chain INH-NAD tight-binding inhibitor. The predictions of ligand-protein interactions at the molecular level can be of primary importance in elucidating the mechanisms of action of isoniazid and InhA-related resistances, in identifying the effective mycobactericidal entities and, in further step, in the design of a new generation of antitubercular drugs.

International audience; Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone-type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure-InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.

Simplified analogues of oxidized and reduced isoniazid–NAD(P) adducts were prepared to study their behaviour with regard to ring–chain tautomeric isomerism in solution. In DMSO, the oxidized analogues (pyridinium salts) and the corresponding 1,2-dihydropyridine-reduced compounds were found to exist exclusively in the ring (cyclic hemiamidal) form. In contrast, the 1,4-dihydropyridine-reduced analogues were present in the ring and/or chain forms depending on the nature of the aromatic substituent. The 1,4-dihydropyridines (Ar = Ph, 3Cl-Py) are, in solution, preferentially in the keto–amide chain form, whereas the pyridine analogue (Ar = Py), which is the closest model of the isoniazid–NAD(P) adduct, exists as ring (major) and chain (minor) tautomers in equilibrium. The ratio of the tautomeric forms involved in the equilibrium of this system is also influenced by the polarity of the solvent with a shift towards the ring tautomer when the polarity of the solvent is increased. Complementary computational studies were performed by using quantum chemical calculations (B3LYP/6-31G**) and frontier molecular orbital analysis, which allowed the key structural factors involved in the ring–chain tautomerism equilibrium to be discussed. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

International audience; InhA is an enoyl-ACP reductase of Mycobacterium tuberculosis implicated in the biosynthesis of mycolic acids, essential constituents of the mycobacterial cell wall. To date, this enzyme is considered as a promising target for the discovery of novel antitubercular drugs. In this work, we describe the first crystal structure of the apo form of the wild-type InhA at 1.80 angstrom resolution as well as the crystal structure of InhA in complex with the synthetic metabolite of the antitubercular drug isoniazid refined to 1.40 angstrom. This metabolite, synthesized in the absence of InhA, is able to displace and replace the cofactor NADH in the enzyme active site. This work provides a unique opportunity to enlighten the structural adaptation of apo-InhA to the binding of the NADH cofactor or of the isoniazid adduct. In addition, a differential scanning fluorimetry study of InhA, in the apo-form as well as in the presence of NAD(+), NADH and INH-NADH was performed showing that binding of the INH-NADH adduct had a strong stabilizing effect.

An overview of the biomimetic catalysts used in the oxidative activation of drugs is given, with an emphasis on the use of synthetic metalloporphyrins as models of cytochrome P450 to mimic the in vivo metabolism of pharmaceuticals. In addition, a special focus is directed towards the recent results from the authors' group on the oxidative activation of isoniazid, an antitubercular drug.

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 Mn III -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.

Isoniazid (INH) is easily oxidized with manganese(III) pyrophosphate, a chemical model of the KatG protein involved in activation of INH inside the bacteria Mycobacterium tuberculosis. Performed in the presence of NAD(+), this oxidation generates a family of isomeric INH-NAD(H) adducts, which have been shown to be effective inhibitors of InhA, an enzyme essential in mycolic acid biosynthesis. In this work, we fully characterized by (1)H and (13)C NMR spectroscopy four main species of INH-NAD(H) adducts that coexist in solution. Two of them are open diastereoisomers consisting of the covalent attachment of the isonicotinoyl radical at position four of the nicotinamide coenzyme. The other two result from a cyclization involving the amide group from the nicotinamide and the carbonyl group from the isonicotinoyl radical to give diastereoisomeric hemiamidals. Although an INH-NAD(H) adduct with a 4S configuration has been characterized within the active site of InhA from Xray crystallography and this bound adduct interpreted as an open form (Rozwarski et al., Science 1998, 279, 98-102), it is legitimate to raise the question about the effective active form(s), open or cyclic, of INH-NAD(H) adduct(s). Is there a single active form or are several forms able to inhibit the InhA activity with different levels of inhibitory potency?

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.

The antituberculosis drug isoniazid (INH) is quickly oxidised by stoichiometric amounts of manganese(III)-pyrophosphate and the following products were identified: isonicotinic acid 1 , isonicotinamide 2 and isonicotinaldehyde 3 , the acid being the major product. The oxidation of INH with Mn III -pyrophosphate was carried out in either H 2 16 O, or H 2 18 O or D 2 O and under varied atmosphere composition (argon, air, O 2 or 18 O 2 ). LC–MS analyses of isotope incorporation suggest the simultaneous presence of two competitive pathways leading to the formation of acid 1 , with the isonicotinoyl radical as a common intermediate. One route is oxygen-dependent and the other is water-dependent. Analyses of isotope incorporation in amide 2 and aldehyde 3 also support this mechanism.

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.

It is currently believed that isoniazid (INH) is oxidised inside Mycobacterium tuberculosis to generate, by covalent attachment to the nicotinamide ring of NAD(H) (beta-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/Mn(II)-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.

The 5'-aldehyde terminus is a DNA oxidative damage resulting from attack at C5' of 2-deoxyriboses by some potent natural or chemical DNA cleavers. To offer a fast and specific method for characterization of this type of damage, we used on-line electrospray ionization mass spectrometry (ESI-MS) detection during liquid chromatography analyses. The intrinsic reactivity of 5'-aldehyde terminus with nucleophiles (formation of hydrate with water, of a Tris adduct with Tris buffer) or through beta-elimination reaction resulted in complex LC profiles and MS data. We showed that derivatization of the aldehyde function as an oxime ether gives a stable derivative easy to characterize during on-line ESI-MS analyses. Complete structural characterization of the Tris adduct and the oxime ether derivative were obtained from MS and detailed NMR studies performed on derivatized 5'-aldehyde thymidine models.

The structure of the title compound 2 , catena -poly{aqua(dimethylformamide- O )bis{μ-[bis( p -nitrophenyl)phosphato]}zinc(II) mono(dimethylformamide)}, consists of linear polymers in which adjacent Zn 2+ cations are joined by the oxygen atoms of two bidentate bridging phosphate diester ligands 1 . The metal shows an octahedral coordination geometry with the zinc connected to six O atoms, four belonging to four different surrounding phosphate groups, one to a water molecule and one to a DMF molecule. Each single-strand is built-up from Zn octahedrons having alternate configuration Δ or Λ.

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.

In contrast to the abundant literature available on the oxidation of primary and secondary alcohols, examples of oxidation of tertiary alcohols are rather rare and a general review on this topic does not exist. Therefore, this microreview will briefly describe the different existing methods, mainly metal-mediated, which consist of one or two-electron oxidations and usually lead to rearrangement, cyclization or fragmentation products. Some related data is also given for fragmentation reactions observed in basic or acid medium, or catalyzed by metal complexes without a real oxidation step.

Oxidation of 4-(1-hydroxy-1-phenylethyl)benzoic acid HPEBA with a water-soluble metalloporphyrin as catalyst and KHSO5 as oxygen atom donor gives the major products, acetophenone AC and acetylbenzoic acid ABA, by a Caliph–CAr bond cleavage, but a minor product, benzoyloxybenzoic acid BOBA, requires the insertion of an oxygen atom to form the ester. This compound becomes the main oxidation product on increasing the amount of acetonitrile in the reaction medium, and its formation is oxygen-dependent. The conversion is drastically lowered by using D2O instead of H2O, suggesting that an alkoxyl radical is formed in the rate-determining step. Labeling experiments using 18O2 or H218O under different reaction conditions show that the carbonyl oxygen atoms of AC and ABA originate either from substrate, water or dioxygen. However, the carbonyl oxygen atom in the ester group of BOBA originates from dioxygen while the other oxygen atom of the ester remains unlabeled. These results can be explained by an O-neophyl rearrangement of the initial alkoxyl radical to afford a carbon-based radical which then reacts with dioxygen or MnIV-OH/water. In a competitive reaction pathway, direct β-scission of the alkoxyl radical leads to unlabeled products. The oxidation of other tertiary diaryl alcohols is also discussed.

Autoxidation of sulfite is catalyzed by the water-soluble complex (meso-tetrakis(4-N-methylpyridiniumyl)porphyrinato)manganese(III) (Mn-TMPyP), leading to an intermediate species capable of hydroxylation of p-isopropylbenzoic acid, epoxidation of carbamazepine, and generation of single-strand breaks both in plasmid ΦX174 DNA and in a 167-base-pair fragment of pBR322. 18O-Labeling studies confirm that the high-valent manganese−oxo intermediate undergoes oxo−hydroxo tautomerism in competition with oxidation of substrate or reduction by excess sulfite. In contrast to the classical reductive activation of dioxygen by metalloporphyrins (dithiothreitol or ascorbate in the presence of O2), a mechanism is proposed in which SO3•-, formed by oxidation with MnIII, is trapped by O2, leading to a species (SO5•-) capable of generating a MnVO complex. Importantly, these studies point to the use of an alternative, biocompatible oxidation system compared to the preformed oxidant KHSO5.

The iron complexes of a new series of weakly acidic acetamidosulfonylated tetraarylporphyrins were tested in aqueous medium as biomimetic oxidation catalysts of the usual peroxidase substrates; tetramethylbenzidine, guaiacol and ABTS. One of them ( meso -tetrakis[4-(acetamidosulfonyl)phenyl]porphyrinato-iron(III), activated by potassium monopersulfate, was shown to be much more efficient than the iron complexes of sulfonated tetraarylporphyrins and at least as efficient as horseradish peroxidase/H 2 O 2 used as reference system. The biomimetic oxidation of acetaminophen is presented as a convenient application of such chemical catalysts in oxidative drug metabolism studies.

High valent metal–oxo species have been evoked as active intermediates in many different oxidation reactions using manganese or iron porphyrin complexes as catalysts and oxygen atom donors (H2O2, PhIO, NaOCl, KHSO5, ... etc.) or dioxygen associated to a reductant as oxygen atom source. When these metalloporphyrin-catalysed oxidations are performed in water, such metal–oxo species are able to transfer an oxygen atom coming from either the oxygen source or from bulk water. This fact has been explained by the so-called oxo–hydroxo tautomerism, a mechanism involving a rapid shift of two electrons and one proton from a hydroxo ligand (electron-rich ligand formed by deprotonation of an aqua ligand) to the trans oxo species (electron-poor ligand) leading to the transformation of the hydroxo ligand into an electrophilic oxo entity on the opposite side of the initial oxo. This ‘oxo–hydroxo tautomerism’, evidenced by using 18O-labelled water, has been used as mechanistic tool to unambiguously characterize oxygen atom transfer mechanisms mediated by metal–oxo species in opposition to mechanisms related to free radical oxidation reactions.

The preparation of tetracationic metalloporphyrin precursors for the attachement to DNA binding molecules is reported. Based on a D4h tetrapyridylporphyrin, such precursors are more accessible than the classical tailored tricationic porphyrin derivatives used as DNA cleavers. meso-Tetrapyridylporphyrin was reacted with ethyl bromopentanoate and methyl iodide to afford a monofunctionalized ester derivative of 1 which was hydrolyzed to the corresponding acid 5. This acid was reacted with spermine and BOP to yield conjugates containing one (6) or two (7) porphyrin units that were metalated with Mn(II) salts to afford the metalloporphyrins 6−Mn and 7−Mn. Furthermore, 6−Mn was linked to 5 to yield a spermine conjugate 8 containing two porphyrin moieties, one of which was metalated. The DNA cleavage activity of these different metalloporphyrins was studied in the presence of KHSO5 with double-stranded ΦX174 DNA. Conjugate 5−Mn, the spermine containing compound 6−Mn, and compound 7−Mn which contained two porphyr...

International audience; The metalloporphyrin-catalyzed oxidation of SSR180575, a ligand of the peripheral benzodiazepine receptor, that contains both N-methylindole and N,N-dimethylamide functions, produces a high yield of a stable carbinolamide derivative.; Le composé SSR180575 est un ligand du récepteur périphérique des benzodiazépines, qui contient à la fois un cycle N-méthylindole et une chaîne N,N-diméthylamide. L’oxydation de ce composé catalysée par des métalloporphyrines produit avec un bon rendement un carbinolamide stable.

MEDLINE AN 2013883009(Journal; Article; (JOURNAL ARTICLE)); Isoniazid (INH) is one of the most commonly used drugs in treatment of human tuberculosis and the most efficient. Although it has been 60 years since isoniazid was introduced in anti-tubercular therapy and despite the simplicity of its chemical structure (C H N O) with few functional groups, its exact mechanism of action, which could account for its specificity and exceptional potency against Mycobacterium tuberculosis and justify all profiles of INH-resistance, remains elusive and debatable. This complexity can find an explanation in the high reactivity of INH and also in the possibility that multiple targets and pathways could co-exist for this medicinal agent. Indeed, since the discovery of isoniazid's anti-tubercular potency, several propositions for its mode of action have been reported, including its conversion, by a catalase peroxidase within M. tuberculosis, into an active metabolite able, after reaction with NAD, to inhibit an enzyme (InhA) crucial to M. tuberculosis survival. This represents the most consensual mechanism described to date. Nevertheless, none of the proposed mechanisms considered independently can explain the singular and privileged action of the isoniazid structure on the tubercle bacillus, or all the profiles of resistance. The aim of this paper is to reconsider the literature reporting the different modes of action described for isoniazid in the light of the present and most relevant knowledge, with special attention to understanding the molecular mechanistic aspects of the drug's action.[on SciFinder (R)]

This review article focuses on the molecular aspects of DNA cleavage by synthetic chemical nucleases (transition metal complexes endowed with redox properties and DNA affinity) and natural drugs (cytotoxic agents such as bleomycins or enediynes). Unlike deoxyribonucleases, which catalyze the nucleophilic attack of water on the phosphorus atom of a particular phosphodiester entity, these nonhydrolytic DNA-cleavers are able to oxidize the sugar units, generally by hydrogen atom abstraction. Examples of oxidative attack on each of the five different CH bonds of deoxyribose are known, depending on the nature, structure, type of activation, or mode of DNA interaction of the DNA-cleaver. Further evolution at the site of the initial lesion leads to the release of bases, oxidized deoxyribose units, or oxidized sugar fragments appended to the base or the terminal phosphate. In most cases the loss of a part (at least) of a nucleoside, with the concomitant loss of one base information, primarily induces the cleavage of the DNA strand. For both types of DNA cleavage reagents studied within the two last decades, the modes of activation and DNA binding are presented, as well as the details on the mechanism of deoxyribose oxidative degration. Because of the need for highly efficient and highly specific reagents, the development of new artificial and selective DNA cleavers, supported by an improved knowledge of these different mechanisms of DNA cleavage, is to-day a challenging area in the rational design of antitumoral or antiviral agents, as well as in the field of molecular biology.

Diese Ubersicht befast sich mit den molekularen Aspekten der DNA-Spaltung durch synthetische chemische Nucleasen (Ubergangsmetallkomplexe mit Redoxeigenschaften und Affinitat zu DNA) und naturliche Wirkstoffe (cytotoxische Agentien wie Bleomycine und Endiine). Anders als die Desoxyribo-Nucleasen, die den nucleophilen Angriff von Wasser auf das Phosphoratom einer bestimmten Phosphodiester-Einheit katalysieren, oxidieren diese nicht hydrolytisch wirkenden Reagentien zur DNA-Spaltung die Zuckereinheiten, und zwar im allgemeinen durch H-Abstraktion. Je nach der Art und der Struktur des Spaltungsreagens sowie der Art seiner Aktivierung und seiner Intercalation in DNA wird eine andere der funf aktivierbaren CH-Bindungen der Desoxyribose angegriffen. In Folgereaktionen an der ursprunglichen Schadigungsstelle werden Nucleobasen freigesetzt, Zuckerreste oxidiert, Basen-substituierte oxidierte Zucker oder an die endstandige Phosphatgruppe gebundene oxidierte Zucker gebildet. In den meisten Fallen verursacht vor allem der Verlust eines Nucleosids oder zumindest eines Teils davon mit dem damit einhergehenden Verlust einer Baseninformation den DNA-Strangbruch. Fur beide Arten von DNA-Spaltungsreagentien wird hier der derzeitige Wissensstand hinsichtlich der Art der Aktivierung und der DNA-Bindung sowie des Mechanismus des oxidativen Desoxyribose-Abbaus vorgestellt. Dank eines genaueren Verstandnisses der unterschiedlichen DNA-Spaltungs-mechanismen und wegen der Nachfragenach hocheffizienten und hochspezifischen Reagentien scheint die Entwicklung neuer kunstlicher und selektiver DNA-Spaltungsreagentien ein herausforderndes Arbeitsgebiet auf dem Wegzu einem rationalen Design neuartiger Antitumor-und antiviraler Agentien sowie in der Molekularbiologie zu sein.

Five lipophilic analogues 1-5 of the active metabolite of the antitubercular drug isoniazid (INH), selected as inhibitors of Mycobacterium smegmatis and Mycobacterium tuberculosis growth, were evaluated for their activity against Corynebacterium glutamicum (lacking in InhA activity), Escherichia coli (to test mycobacteria selectivity), and Plasmodium falciparum (as possible parasite target). Compound 3 was the only one that did not inhibit C. glutamicum growth. The poor InhA inhibitors 1 and 2 were able to inhibit C. glutamicum and their anti(myco)bacterial mechanisms of action involve targets other than InhA. For the effective InhA inhibitors 4 and 5, also active against C. glutamicum and M. tuberculosis strains, more than one pathway should be envisaged to explain their actions. Pyridine-base ring analogues (1, 2, and 3) have no ability to inhibit the growth of E. coli even at a high concentration. Compound 3 thus exhibited a selective inhibitory action toward M. tuberculosis, while it was inactive on C. glutamicum and on E. coli growth. It presented an activity profile similar to that of INH suggesting InhA inhibition as one of the possible mechanisms of action. Finally, although a homologue of the reductase InhA exists in the FAS-II system of P. falciparum, 3 was unable to display antiplasmodial activity.

The synthesis and biological evaluation of azaisoindolinone compounds embedding a lipophilic chain on the framework were performed. These compounds were designed as InhA inhibitors and as anti-Mycobacterium tuberculosis agents. Structure-activity relationships concerning the length and the location of the lipophilic chain around the azaisoindolinone framework, the suppression of the phenyl group, the bioisosteric substitution of ether link and alkylating of the tertiary hydroxyl and the hemiamidal nitrogen were also investigated, revealing insightful information and thereby enabling further diversification of the azaisoindolinone scaffold for new antitubercular agents.

Photochemotherapy is used both for solid tumors and in extracorporeal treatment of various hematologic disorders. Nevertheless, its development in oncology remains limited, because of the low selectivity of photosensitizers (PS) towards human tumor cells. To enhance PS efficiency, we recently covalently linked a porphyrin (TrMPyP) to a plant lectin (Morniga G), known to recognize with high affinity tumor-associated T and Tn antigens. The conjugation allowed a quick uptake of PS by Tn-positive Jurkat leukemia cells and efficient PS-induced phototoxicity. The present study was performed: (i) to evaluate the targeting potential of the conjugate towards tumor and normal cells and its phototoxicity on various leukemia cells, (ii) to investigate the mechanism of conjugate-mediated cell death. The conjugate: (i) strongly increased (x1000) the PS phototoxicity towards leukemic Jurkat T cells through an O-glycan-dependent process; (ii) specifically purged tumor cells from a 1:1 mixture of Jurkat leukemia (Tn-positive) and healthy (Tn-negative) lymphocytes, preserving the activation potential of healthy lymphocytes; (iii) was effective against various leukemic cell lines with distinct phenotypes, as well as fresh human primary acute and chronic lymphoid leukemia cells; (iv) induced mostly a caspase-independent cell death, which might be an advantage as tumor cells often resist caspase-dependent cell death. Altogether, the present observations suggest that conjugation with plant lectins can allow targeting of photosensitizers towards aberrant glycosylation of tumor cells, e. g. to purge leukemia cells from blood and to preserve the normal leukocytes in extracorporeal photochemotherapy.

The chemical nuclease Mn-TMPyP/KHSO 5 is able to hydroxylate 5' carbon-hydrogen bonds of deoxyribose units at three contiguous A-T base pairs sites and initiate DNA rupture. Double-strand cleavage sites consist of trinucleotide sequences with a four nucleotide 3' stagger of the cleaved residues. Both strand nicks are identical and consist of 3'-phosphate termini facing a 5'-aldehyde residue which is easily reduced to a 5'-alcohol by NaBH 4 . These two successive treatments (oxidation plus reduction) are equivalent to a hydrolysis of the phosphodiester bond. Such mechanism is reminiscent of DNA restriction enzymes but with 5'-OH and 3'-phosphate ends at the site of cleavage

This review article focuses on the molecular aspects of DNA cleavage by synthetic chemical nucleases (transition metal complexes endowed with redox properties and DNA affinity) and natural drugs (cytotoxic agents such as bleomycins or enediynes). Unlike deoxyribonucleases, which catalyze the nucleophilic attack of water on the phosphorus atom of a particular phosphodiester entity, these nonhydrolytic DNA-cleavers are able to oxidize the sugar units, generally by hydrogen atom abstraction. Examples of oxidative attack on each of the five different CH bonds of deoxyribose are known, depending on the nature, structure, type of activation, or mode of DNA interaction of the DNA-cleaver. Further evolution at the site of the initial lesion leads to the release of bases, oxidized deoxyribose units, or oxidized sugar fragments appended to the base or the terminal phosphate. In most cases the loss of a part (at least) of a nucleoside, with the concomitant loss of one base information, primarily induces the cleavage of the DNA strand. For both types of DNA cleavage reagents studied within the two last decades, the modes of activation and DNA binding are presented, as well as the details on the mechanism of deoxyribose oxidative degration. Because of the need for highly efficient and highly specific reagents, the development of new artificial and selective DNA cleavers, supported by an improved knowledge of these different mechanisms of DNA cleavage, is to-day a challenging area in the rational design of antitumoral or antiviral agents, as well as in the field of molecular biology.

This review article presents the different active species involved in the oxidative DNA cleavage by chemical nucleases and drugs: ionizing radiation, reduced oxygen species, singlet oxygen, peroxides, excited states of photosensitizers, organic radicals and high-valent metal-oxo entities

DNA damage photoinduced by four nonsteroidal antiinflammatory drugs (NSAID) have been investigated by neutral agarose gel electrophoresis. Upon irradiation at 300 nm, in phosphate buffered solution, benoxaprofen, naproxen, ketoprofen, tiaprofenic acid photosensitized the formation of single-strand breaks (SSB) in double stranded supercoiled phi X174 DNA. The efficiency of the cleavage is higher in argon saturated solutions than in aerated solutions and it is not correlated with the quantum yield of photodegradation of the drugs. Simultaneously with the DNA strand breaks, NSAID promote a weak reduction of the electrophoretic mobility of the supercoiled form that may be attributed to the formation of pyrimidine dimers or other DNA unwinding products. These photodimerization processes suggest the involvement of a triplet-triplet energy transfer between NSAID and DNA. Addition of mannitol and superoxide dismutase decreases the efficiency of the cleavage suggesting that HO. and O2.- are involved in the DNA cleavage. Unexpectedly, addition of sodium azide quenches the cleavage both in aerated or in deaerated solutions. Substituting H2O by D2O does not change the number of SSB thus suggesting that 1O2 does not take an important place in the cleavage of DNA. From our data we tentatively assume that the cleavage occurs through a radical mechanism that may involve in a first step an energy or an electron transfer. Gel sequencing on NSAID-photoinduced DNA breakage exhibits no particular specificity except in the case of benoxaprofen where a slight selectivity for cytosine is observed.

Mn(III) and Fe(III) complexes of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (M-TMePyP) and related hybrid molecules ("metalloporphyrin-ellipticine") were activated by potassium monopersulfate in the presence of variable calf thymus (CT) DNA and NaCl concentrations. Monitored by visible spectroscopy (Soret band), fast degradation of the free metalloprophyrin was observed while the DNA-bound form appeared protected. This direct quantitation of free versus bound metalloporphyrin ratios allowed determination of binding constants: Mn- and Fe-TMePyP respectively bind to CT DNA (5 mM phosphate buffer, 0.1 M NaCl, pH 7) with K = 3 X 10(4) and 1.2 X 10(4) M-1. Mn-TMePyP showed a greater affinity for poly[d(A-T)] (K = 1.2 X 10(5) M-1) than for poly[d(G-C)] (K = 0.2 X 10(4) M-1). This method allowed us access to the intrinsic DNA affinity of the metalloporphyrin moiety of the hybrid molecules "metalloporphyrin-ellipticine".

Phosphorus-31 NMR has been applied to the characterization of terminal phosphates on fragments of calf thymus DNA induced by three different nuclease systems: DNase I, DNase II and the artificial nuclease 'Mn-TMPyP/KHSO5'. In this last case, the oxidative damage to deoxyribose leads to two monophosphates esters (at the 3' and 5' ends) on both sides of the cleavage site. This method constitutes a promising approach to visualise the phosphate termini generated in DNA or RNA cleavage by cytotoxic drugs or chemical nucleases and provides a novel insight into the molecular aspects of their mechanism of action.

Mn TMPyP in the presence of sulfite/O2 catalyses the oxidation of dG into dIz as selectively but slower and less efficiently than in the presence of KHSO5.

Esters of elliptinium with stearic (ST-NME), palmitic (PAL-NME) or oleic (OL-NME) acids, a series of lipophilic derivatives of ellipticine, were synthetized, in order to evaluate their incorporation into Low Density Lipoprotein (LDL). Among the three derivatives, OL-NME shows the most potent incorporation (83 micrograms/mg protein LDL) compared to ST-NME (37 micrograms/mg protein LDL) and PAL-NME (58 micrograms/mg protein LDL). The size of OL-NME-LDL was determined by size distribution particles, showing their homogeneity compared to native LDL. When culture normal human fibroblasts were incubated with [125I]LDL incorporated drug, they bound to the LDL receptor with the same affinity as native LDL and were internalized and degraded intracellularly. The presence of excess native LDL inhibited the cellular uptake and degradation of [125I]drug-LDL. We have used [125I]acetyl-LDL as a probe for a binding site on macrophages that mediated the uptake and degradation of chemically altered or denatured LDL. Mouse peritoneal macrophages were shown to take up and degrade [125I]acetyl-LDL at rates that were greater than those for the uptake and degradation of native [125I]LDL and [125I]drug-LDL. The in vitro cytotoxic test on L1210 murine leukemic cells demonstrated that the complex was cytotoxic and was more effective than the free drug. This cytotoxic activity of the drug-LDL complex depends on the LDL high affinity receptor since the addition of native LDL reduces the killing power. In contrast, methylated LDL, which does not bind to the LDL receptor, has no effect on it. We conclude that it is possible to incorporate a large amount of cytotoxic drug into LDL without modifying their cellular metabolism via the high affinity LDL receptor pathway. It indicates also that the delivery of lipophilic drugs using LDL might provide distinct advantages over the use of synthetic carriers.

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.

[reaction: see text] An isoniazid-NAD adduct has been recently proposed as the ultimate metabolite responsible for the antituberculous activity of isoniazid (INH). Its structure results from binding of the isonicotinoyl radical at C4 position of the nicotinamide ring of NAD with further possible and debated cyclization to form a cyclic hemiamidal derivative. Replacing the pyridine cycle of INH in INH-NAD adduct by a phenyl cycle (BH-NAD adduct) was shown previously to still retain the activity. On these bases, the core structure (4-benzoyl-1,4-dihydronicotinamide ribonucleoside) of the BH-NAD adduct and a series of analogues have been synthesized by using 3,4-pyridinedicarboximide as starting material. Depending on the nature of the substituent (pyridine or aryl) and on the oxidized or the reduced state of the nicotinamide nucleus, they were found either in a cyclized hemiamidal or an opened form or were shown to exist in equilibrium under cyclized or opened forms. Although none of these compounds could significantly inhibit activity of the InhA or MabA reductases (two possible targets of isoniazid), they represent attractive targets to develop potential second-generation inhibitors, including the total chemical synthesis of the bioactive BH-NAD adduct.

Oxidative activation of the antituberculous drug isoniazid (INH) in the presence of the NADH cofactor gives a pool of INH–NAD adducts proposed to be involved in the mechanism of action of this drug through inhibition of the reductase InhA. Among these adducts and besides dihydropyridine derivatives, two pyridinium-type isoniazid–NAD adducts were shown to be formed in solution and have been fully characterized by 1H/13C NMR and MS. One of them results from the oxidation of dihydropyridine-type INH–NAD adducts. The spectral data strongly support its existence under two epimeric structures. These epimers arise from a cyclization process between the carboxamide group and the ketone function with the creation of a new chiral center at C-7. The second pyridinium-type adduct was formed in acidic solution by dehydration of the cyclized dihydropyridine-type INH–NAD adducts and also exists as a cyclized structure. Both of these pyridinium-type compounds were inactive as inhibitors of InhA activity and can be considered as deactivated species.

An ortho-metallation-electrophilic substitution sequence was employed as a key step to build the 4-benzoylpyridine framework. It was found that 4-benzoylpyridine-3-carboxamide and an N-pyridyl alkylated derivative both exist in a unique cyclized hemiamidal structure, not in the usually expected keto-amide open form. These structures represent fragment models of the Isoniazid-NAD adducts involved in the mechanism of action of the antituberculous drug Isoniazid.

Hydroxylation of poly(dA)·poly(dT) at the 5′ position of the deoxyribose units by the chemical nuclease Mn-TMPyP–KHSO5[Mn–TMPyP = manganese(III) complex of meso-tetrakis(4-N-methylpyridiniumyl)porphyrin] followed by an additional thermal step provides 5′-aldehyde-3′,4′-unsaturated nucleosides which can be reduced by sodium borohydride and then hydrogenated with palladium on charcoal giving 2′,3′-dideoxyribonucleosides having the natural β or the non-natural α configuration at 4′.

In contrast to the abundant literature available on the oxidation of primary and secondary alcohols, examples of oxidation of tertiary alcohols are rather rare and a general review on this topic does not exist. Therefore, this microreview will briefly describe the different existing methods, mainly metal-mediated, which consist of one or two-electron oxidations and usually lead to rearrangement, cyclization or fragmentation products. Some related data is also given for fragmentation reactions observed in basic or acid medium, or catalyzed by metal complexes without a real oxidation step.