Michel Nguyen

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.

Regulation of Copper and Iron Homeostasis by Metal Chelators: A Possible Chemotherapy for Alzheimer’s Disease

With the increase of life expectancy of humans in more than two-thirds of the countries in the World, aging diseases are becoming the frontline health problems. Alzheimers disease (AD) is now one of the major challenges in drug discovery, since, with the exception of memantine in 2003, all clinical trials with drug candidates failed over the past decade. If we consider that the loss of neurons is due to a high level of oxidative stress produced by nonregulated redox active metal ions like copper linked to amyloids of different sizes, regulation of metal homeostasis is a key target. The difficulty for large copper-carrier proteins to directly extract copper ions from metalated amyloids might be considered as being at the origin of the rupture of the copper homeostasis regulation in AD brains. So, there is an urgent need for new specific metal chelators that should be able to regulate the homeostasis of metal ions, specially copper and iron, in AD brains. As a consequence of that concept, chelators promoting metal excretion from brain are not desired. One should favor ligands able to extract copper ions from sinks (amyloids being the major one) and to transfer these redox-active metal ions to copper-carrier proteins or copper-containing enzymes. Obviously, the affinity of these chelators for the metal ion should not be a sufficient criterion, but the metal specificity and the ability of the chelators to release the metal under specific biological conditions should be considered. Such an approach is still largely unexplored. The requirements for the chelators are very high (ability to cross the brain-blood barrier, lack of toxicity, etc.), few chemical series were proposed, and, among them, biochemical or biological data are scarce. As a matter of fact, the bioinorganic pharmacology of AD represents less than 1% of all articles dedicated to AD drug research. The major part of these articles deals with an old and rather toxic drug, clioquinol and related analogs, that do not efficiently extract copper from soluble amyloids. We have designed and developed new tetradendate ligands such as 21 and PA1637 based on bis(8-aminoquinolines) that are specific for copper chelation and are able to extract copper(II) from amyloids and then can release copper ion upon reduction with a biological reducing agent. These studies contribute to the understanding of the physicochemical properties of the tetradentate copper ligands compared with bidentate ligands like clioquinol. One of these copper ligands, PA1637, after selection with a nontransgenic mouse model that is able to efficiently monitor the loss of episodic memory, is currently under preclinical development.

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.

Characterization of new specific copper chelators as potential drugs for the treatment of Alzheimer's disease.

The non-controlled redox-active metal ions, especially copper, in the brain of patients with Alzheimer disease (AD) should be considered at the origin of the intense oxidative damage in the AD brain. Several bis(8-aminoquinoline) ligands, such as 1 and PA1637, are able to chelate Cu(2+) with high affinity, and are specific chelators of copper with respect to iron and zinc. They are able to efficiently extract Cu(2+) from a metal-loaded amyloid. In addition, these tetradentate ligands are specific for the chelation of Cu(2+) compared with Cu(+). Consequently, the copper ion is easily released from the bis(8-aminoquinoline) ligand under reductive conditions, and can be trapped again by a protein having some affinity for copper such as human serum albumin (HSA) proteins. In addition, the copper is not efficiently released from [Cu(CQ)2] in reductive conditions. The catalytic production of H2O2 by [Cu(2+)-Aβ(1-28)]/ascorbate is inhibited in vitro by the bis(8-aminoquinoline) 1, suggesting that 1 should be able to play a protective role against oxidative damages induced by copper-loaded amyloids.

The Necessity of Having a Tetradentate Ligand to Extract Copper(II) Ions from Amyloids

The accumulation of redox-active metal ions, in particular copper, in amyloid plaques is considered to the cause of the intensive oxidation damage to the brain of patients with Alzheimers disease (AD). Drug candidates based on a bis(8-aminoquinoline) tetradentate ligand are able to efficiently extract Cu2+ from copper-loaded amyloids (Cu–Aβ). Contrarily, in the presence of a bidentate hydroxyquinoline, such as clioquinol, the copper is not released from Aβ, but remains sequestrated within a Aβ–Cu–clioquinol ternary complex that has been characterized by mass spectrometry. Facile extraction of copper(II) at a low amyloid/ligand ratio is essential for the re-introduction of copper in regular metal circulation in the brain. As, upon reduction, the Cu+ is easily released from the bis(8-aminoquinoline) ligand unable to accommodate CuI, it should be taken by proteins with an affinity for copper. So, the tetradentate bis(8-aminoquinoline) described here might act as a regulator of copper homeostasis.

Transfer of Copper from an Amyloid to a Natural Copper‐Carrier Peptide with a Specific Mediating Ligand

The oxidative stress that arises from the catalytic reduction of dioxygen by Cu(II/I)-loaded amyloids is the major pathway for neuron death that occurs in Alzheimers disease. In this work, we show that bis-8(aminoquinoline) ligands, copper(II) specific chelators, are able to catalytically extract Cu(II) from Cu-Aβ1-16 and then completely release Cu(I) in the presence of glutathione to provide a Cu(I)-glutathione complex, a biological intermediate that is able to deliver copper to apo forms of copper-protein complexes. These data demonstrate that bis-8(aminoquinolines) can perform the transfer of copper ions from the pathological Cu-amyloid complexes to regular copper-protein complexes. These copper-specific ligands assist GSH to recycle Cu(I) in an AD brain and consequently slow down oxidative damage that is due to copper dysregulation in Alzheimers disease. Under the same conditions, we have shown that the copper complex of PBT2, a mono(8-hydroxyquinoline) previously used as a drug candidate, does not efficiently release copper in the presence of GSH. In addition, we report that GSH itself was unable to fully abstract copper ions from Cu-β-amyloid complexes.

Is the isonicotinoyl radical generated during activation of isoniazid by MnIII-pyrophosphate?

Abstract 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.

The nonenzymatic activation of isoniazid by MnIII-pyrophosphate in the presence of NADH produces the inhibition of the enoyl-ACP reductase InhA from Mycobacterium tuberculosis

Abstract The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III)-pyrophosphate. In the presence of the nicotinamide coenzyme, the INH oxidation produced the formation of INH-NAD(H) adducts and allowed the in vitro inhibition of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway for mycolic acids. Manganese(III)-pyrophosphate is an efficient 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 on reactive INH species and resulting InhA inhibitors.

Preparation of Tetradentate Copper Chelators as Potential Anti‐Alzheimer Agents

The uncontrolled redox activity of metal ions, especially copper, in the brains of patients with Alzheimers disease (AD) should be considered the origin of intense oxidative damage to neurons in the AD brain. To obtain low‐molecular‐weight copper chelators that act as tetradentate ligands, we designed new compounds based on an 8‐aminoquinoline motif with a lateral chain attached at the 2‐position of the aromatic ring. Some of these new ligands, termed TDMQ for TetraDentate MonoQuinolines, are specific for copper chelation. Full characterization of these ligands is reported, as well as their affinities for CuII, and their capacities to inhibit oxidative stress induced by copper–amyloids activated by a reductant. Such metal ligands can be considered as potential anti‐AD agents, as they should be able to regulate the homeostasis of copper in brain tissue.