Search

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; 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.

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 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.

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

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.

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.

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′.

To increase the binding affinity and/or the sequence selectivity of the chemical nuclease manganese-(III) tetrakis(4-N-methylpyridiniumyl)porphyrin, we synthesized a conjugate molecule by associating a tris(4-N-methylpyridiniumyl)metalloporphyrin motif to Hoechst 33258 (H33258), a DNA minor groove binding dye known for its selective affinity for A-T tracts. Selected double-stranded (ds) 35-mer oligodeoxyribonucleotides have been used to probe DNA chain breakages induced by the managanese derivative of the conjugate after activation by potassium monopersulfate. Gel electrophoresis analyses show that DNA cuts were generated by the metalloporphyrin moiety of the hybrid molecule, with the H33258 entity interacting in two different possible orientations, upstream or downstream, with its preferred affinity site inside the minor groove. Also studied was the cleavage of a ds 29-mer oligodesoxyribonucleotide containing two stretches of A.T basepair (bp) which clearly showed that the hybrid can occupy the binding region at least in four preferred ways. These cleavage experiments support the strong and selective interaction of the metalloporphyrin-dye hybrid with DNA and allow the estimate of 10 bp as an average size for the affinity site of an isolated conjugate molecule. Further studies by UV-visible spectroscopy, DNA melting temperature determinations, and DNase I footprinting showed, for higher concentrations of H33258 conjugate, a preferential interaction of only the H33258 moiety with DNA (estimated binding site size 6-7 bp) with the porphyrin entity pushed out of the groove and, for the highest concentrations, self-aggregation of the H33258 conjugate all along the DNA strand in a nonselective mode.

Recent experiments with the chemical nuclease Mn-TMPyP/KHSO5 showed highly specific cleavage on both 3′-sides of AT base pair triplets of double stranded oligonucleotides.1 Hydroxylation at the 5′-carbon of a deoxyribose represents the initial damage and leaves a 3′-phosphate and a 5′-aldehyde at the ends. Careful examination of PAGE bands resulting from oxidative cleavage of two selected double-stranded oligodeoxyribonucleotides which have been labeled either at 5′ or 3′ ends indicated that 5′-end-labeled fragments with a 3′-phosphate terminus were unsensitive to different chemical or thermal treatments and migrated according to the corresponding fragments obtained by Maxam-Gilbert sequencing. Whereas 3′-end-labeled fragments with a 5′-aldehyde terminus (-CHO) migrated differently after reduction (-CH2OH), oxidation (-COOH), heating (5′-P terminus and release of free base and furfural), heating and alkaline phosphatase treatment (5′-OH terminus and loss of free base, furfural and inorganic phosphate). The precise knowledge of the chemical lesion at the oxidized site of DNA allowed us to localize the exact position of the break and attribute the structures of the different fragments observed on PAGE analysis after the manganese porphyrin-mediated cleavage of two ODNs containing A.T rich sequences.

Selected double-stranded oligodeoxyribonucleotides have been used to probe, at the molecular level, DNA chain breakages induced by the chemical nuclease mesotetrakis(4-N- methylpyridiniumyl)porphyrinatomanganeseIII pentaacetate/KHSO5. The results show that cleavage selectively occurs on the two 3' sides of three contiguous A.T base pairs (an A.T triplet). Hydroxylation at 5' carbon of the deoxyribose targets represents the initial damage on the sugar-phosphodiester backbone and leaves a 3' phosphate and a 5' aldehyde at the ends. The fragments were separated by HPLC and unambiguously identified through chemical and biochemical reactions and/or sequencing after enzymatic conversion to mononucleosides. Also studied was the degradation of a 22-nucleotide DNA molecule containing two A.T triplets. Gel electrophoresis analyses on the corresponding 5'-32P-end-labeled substrate supported the above cleavage specificity and mechanism.

We report the full structure of two elliptinium diribonucleosides monophosphate adducts: the oxidized form of this antitumor agent alkylates very selectively the pX ribose of ApX (X = G or U) leading to a spiro derivative, where the C10 atom of ellipticine skeleton is linked to both sugar oxygen atoms 2' and 3'. No other adducts could be detected, specially the expected ones corresponding to the usual alkylation sites of bases.

Oxidative cleavage of DNA is observed when the oxygen donor potassium hydrogen persulphate is associated with a water-soluble managanese porphyrin; the nuclease activity is obtained for low concentrations of both the manganese porphyrin (2–200 nM) and potassium hydrogen persulphate (1–25 µM), and the latter is more efficient than hydrogen peroxide in this metalloporphyrin-mediated cleavage of DNA.