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PubMed2001

2- and 3-substituted 1,4-naphthoquinone derivatives as subversive substrates of trypanothione reductase and lipoamide dehydrogenase from Trypanosoma cruzi: synthesis and correlation between redox cycling activities and in vitro cytotoxicity.

Trypanothione reductase (TR) is both a valid and an attractive target for the design of new trypanocidal drugs. Starting from menadione, plumbagin, and juglone, three distinct series of 1,4-naphthoquinones (NQ) were synthesized as potential inhibitors of TR from Trypanosoma cruzi (TcTR). The three parent molecules were functionalized at carbons 2 and/or 3 by various polyamine chains. Optimization of TcTR inhibition and TcTR specificity versus human disulfide reductases was achieved with the 3,3'-[polyaminobis(carbonylalkyl)]bis(1,4-NQ) series 19-20, in which an optimum chain length was determined for inhibition of the trypanothione disulfide reduction. The most active derivatives against trypanosomes in cultures were also studied as subversive substrates of TcTR and lipoamide dehydrogenase (TcLipDH). The activities were measured by following NAD(P)H oxidation as well as coupling the reactions to the reduction of cytochrome c which permits the detection of one-electron transfer. For TcTR, 20(4-c) proved to be a potent subversive substrate and an effective uncompetitive inhibitor versus trypanothione disulfide and NADPH. Molecular modeling studies based on the known X-ray structures of TcTR and hGR were conducted in order to compare the structural features, dimensions, and accessibility of the cavity at the dimer interface of TcTR with that of hGR, as one of the putative NQ binding sites. TcLipDH reduced the plumbagin derivatives by an order of magnitude faster than the corresponding menadione derivatives. Such differences were not observed with the pig heart enzyme. The most efficient and specific subversive substrates of TcTR and TcLipDH exhibited potent antitrypanosomal activity in in vitro T. brucei and T. cruzi cultures. The results obtained here confirm that reduction of NQs by parasitic flavoenzymes is a promising strategy for the development of new trypanocidal drugs.

Animals↗
PubMed1995

Flavoprotein structure and mechanism. 5. Trypanothione reductase and lipoamide dehydrogenase as targets for a structure-based drug design.

Trypanothione reductase (TR) is a flavoenzyme that has been found only in parasitic protozoa of the order Kinetoplastida. The enzyme catalyzes the NADPH-dependent reduction of glutathionylspermidine conjugates and is a key enzyme of the parasite's thiol metabolism. Consequently, TR is an attractive target molecule for a structure-based drug development against Chagas' disease, African sleeping sickness, and other diseases caused by trypanosomes and leishmanias. The three-dimensional structures of TR and of three enzyme substrate complexes have been solved. Several classes of compounds are discussed as guide structures for the design of specific inhibitors. Among them are tricyclic compounds such as acridines and phenothiazines, which competitively inhibit TR but not the related host enzyme glutathione reductase, as well as oxidase activity-inducing quinones and nitrofurans. Lipoamide dehydrogenase (LipDH) is another flavoprotein discussed as a target molecule for an antitrypanosomal therapy. In Trypanosoma cruzi, an organism that is highly susceptible to oxidative stress, LipDH participates in the redox cycling of nifurtimox, one of the most effective anti-Chagas agents. In conclusion, the structurally related enzymes TR and LipDH exhibit an unusually high one-electron-reducing capacity. Consequently, turncoat inhibitors and other compounds inducing an oxidase activity in both enzymes are promising drug candidates against Chagas' disease.

Animals↗
PubMed1995

Mitochondrial NADH-->NAD transhydrogenation in adult Hymenolepis diminuta.

Adult Hymenolepis diminuta mitochondria catalyze a transhydrogenation reaction between NADPH and NAD and between NADH and NAD. The NADPH-->NAD reaction is catalyzed by an inner membrane-associated pyridine nucleotide transhydrogenase, whereas the NADH-->NAD reaction is ostensibly catalyzed by another system(s). The source(s) of NADH-->NAD activity was evaluated by assessments of its intramitochondrial distribution and thermal lability and by comparisons with the distribution/thermal lability of NADH dehydrogenase, lipoamide dehydrogenase, and NADPH-->NAD transhydrogenase. The occurrence of NADH and lipoamide dehydrogenase components was readily demonstrable. Like NADPH-->NAD transhydrogenase, NADH dehydrogenase was essentially membrane bound. Lipoamide dehydrogenase and NADH-->NAD activities were, at different levels, in the membrane and soluble fractions. Based on thermal profiles, NADH and lipoamide dehydrogenase differed from each other and from NADPH-->NAD transhydrogenase. Although the NADH-->NAD profile closely paralleled that for lipoamide dehydrogenase, it also was similar to the NADH dehydrogenase profile. Collectively, these data are consistent with the supposition that the H. diminuta mitochondrial NADH-->NAD transhydrogenation reaction is catalyzed by lipoamide dehydrogenase and possibly by NADH dehydrogenase rather than by an independent transhydrogenase system.

Animals↗
PubMed2001

Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes.

The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD(+) in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C(4)-proS position of the nicotinamide ring are small [(D)(V/K)(NADH) = 1.12 +/- 0.15, (D)V(app) = 1.05 +/- 0.07] when D,L-lipoamide is the oxidant but large and equivalent [(D)(V/K)(NADH) = (D)V = 2.95 +/- 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with (D)(V/K)(APADH) = 0.73 +/- 0.03, (D)(V/K)(Lip(S))2 = 0.77 +/- 0.03, and (D)V(app) = 0.77 +/- 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with (D)(V/K)(APADH) = 0.75 +/- 0.06, (D)(V/K)(DTP) = 0.71 +/- 0.02, and (D)V(app) = 0.56 +/- 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys(46) and Cys(41), limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.

Amino Acid Sequence↗
PubMed1997

Molecular structure of the lipoamide dehydrogenase domain of a surface antigen from Neisseria meningitidis.

The protein p64k from the surface of the Neisseria meningitidis bacteria has been characterized as a two-domain protein. It contains a dihydrolipoamide dehydrogenase domain of 482 residues, involving a FAD prosthetic group as a cofactor, and a smaller lipoic acid binding domain of 86 residues. The two domains are joined by a flexible segment rich in alanine and proline residues. The structure of the dihydrolipoamide dehydrogenase domain was determined by X-ray diffraction. It was solved by a combination of molecular replacement and multiple isomorphous replacement techniques and refined to 2.7 A resolution. In the crystal, the recombinant p64k mimics the functional homo-dimer by using one of the crystallographic 2-fold axes. The reactive disulphide bridge Cys161-Cys166 is in the oxidised state and the FAD is bound in an extended conformation. This main domain contains the major antigenic determinant of the protein, an extended loop of 32 residues at the surface of the protein. A mis-attribution at residue 553 in the sequence has been detected by inspection of electron density maps and the geometry. However, when compared to the other dihydrolipoamide dehydrogenases, there are some significant differences: (1) an unusual number of cis-proline residues and (2) a new motif built around a 2-fold axis by the sulphur atoms of residues Met558, Cys560 and their symmetry related equivalents.

Amino Acid Sequence↗