Detection of DT-diaphorase Enzyme with a ParaCEST MRI Contrast Agent.

A responsive magnetic resonance (MRI) contrast agent has been developed that can detect the enzyme activity of DT-diaphorase. The agent produced different chemical exchange saturation transfer (CEST) MRI signals before and after incubation with the enzyme, NADH, and GSH at different pH values whereas it showed good stability in a reducing environment without enzyme.

Delamanid is not metabolized by Salmonella or human nitroreductases: A possible mechanism for the lack of mutagenicity.

Nitro-containing compounds such as nitrofuran and nitroimidazole are drugs used for the treatment of infectious diseases. However, many of these nitro-containing drugs are positive in the bacterial reverse mutation assay (Ames test). The recently approved anti-multidrug-resistant tuberculosis (MDR-TB) drug, delamanid (Deltyba™; OPC-67683), a derivative of 4-nitroimidazole, was negative for mutagenicity in the Ames assay. In Salmonella typhimurium, mutagenicity of nitro compounds has been closely associated with the ability of nitroreductase to metabolize (degradation)these compounds. To explore the lack of mutagenicity for delamanid, we examined the initial metabolic rate and mutagenic-specific activity of a series of nitro compounds in S. typhimurium TA100. The order of maximum mutagenic-activity was nitrofuran > 2-nitroimidazole > 5-nitroimidazole ≥ 4-nitroimidazole, which is very similar to the order of initial metabolic rate, i.e., the Pearson's correlation coefficient (r = 0.85) showed a correlation between metabolic rate and mutagenic-activity. No metabolism of delamanid was detected even after 60 h of treatment. In addition, delamanid was not reduced by two human nitroreductases. These facts may explain the absence of genotoxicity of delamanid in both in vitro and in vivo tests.

Mitochondrial diaphorases as NAD⁺ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition.

Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD(+) supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD(+) pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD(+) derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain.

Bexarotene prodrugs: targeting through cleavage by NQO1 (DT-diaphorase).

Bexarotene, a retinoid X receptor (RXR) agonist, is being tested as a potential disease modifying treatment for neurodegenerative conditions. To limit the peripheral exposure of bexarotene and release it only in the affected areas of the brain, we designed a prodrug strategy based on the enzyme NAD(P)H/quinone oxidoreductase (NQO1) that is elevated in neurodegenerative diseases. A series of indolequinones (known substrates of NQO1) was synthesized and coupled to bexarotene. Bexarotene-3-(hydroxymethyl)-5-methoxy-1,2-dimethyl-1H-indole-4,7-dione ester 7a was cleaved best by NQO1. The prodrugs are not cleaved by esterase.

Liver Antioxidant Capacity and Steatosis in Laying Hens Exposed to Various Quantities of Lupin (Lupinus angustifolius) Seeds in the Diet.

Despite the many beneficial properties of legume plants, their use in diets for poultry is limited by the presence of antinutritional factors. The aim of the study was to determine the activity of DT-diaphorase, ethoxycoumarin O-deethylase, and catalase, and the concentration of malondialdehyde in liver tissue, as well as the activity of SOD and CAT in the serum of Hy-line Brown hens fed a diet supplemented with various doses of Lupinus angustifolius seeds. The results indicate that the use of large amounts of lupin in the diet resulted in an increase in MDA concentration in the liver and the lipid vacuolization of hepatocytes. A significant increase in DTD activity was observed in chickens receiving 15% lupin. Regardless of lupin dose, no increase in SOD activity was observed in chicken serum after 33 days of the experiment. From the 66th day of the experiment, an increase in catalase activity in the serum of laying hens was observed, while low activity of this enzyme was found in the liver. It can be concluded that the short-term use of lupin in the diet of laying hens does not affect the activity of antioxidant enzymes and, therefore, does not affect the oxidative-antioxidant balance of their body.

Sensitive electrochemical immunosensor via amide hydrolysis by DT-diaphorase combined with five redox-cycling reactions.

Amide hydrolysis using enzyme labels, such as proteases, occurs at a slower rate than phosphoester and carboxyl ester hydrolysis. Thus, it is not very useful for obtaining high signal amplification in biosensors. However, amide hydrolysis is less sensitive to nonenzymatic spontaneous hydrolysis, allowing for lower background levels. Herein, we report that amide hydrolysis by DT-diaphorase (DT-D) occurs rapidly and that its combination with five redox-cycling reactions allows for the development of a highly sensitive electrochemical immunosensor. DT-D rapidly generates ortho-aminohydroxy-naphthalene (oAN) from its amide substrate via amide hydrolysis, which not even trypsin, a highly catalytic protease, can achieve. NADH, which is required for amide hydrolysis, advantageously acts as a reducing agent for rapid electrooxidation-based redox-cycling reactions. In the presence of oAN, DT-D, and NADH, two redox-cycling reactions rapidly occur. In the additional presence of an electron mediator, Ru(NH3)63+ [Ru(III)], three more redox-cycling reactions occur because Ru(III) reacts rapidly with oAN and DT-D. Although the O2-related redox-cycling reactions and redox reaction decrease electrochemical signals, this signal-decreasing effect is not significant in air-saturated solutions. The slow electrooxidation of NADH at an indium tin oxide electrode and sluggish reaction between NADH and Ru(III) allow for low electrochemical backgrounds. When the developed signal amplification scheme is tested for the sandwich-type electrochemical detection of parathyroid hormone (PTH), a detection limit of ∼1 pg/mL is obtained. The detection method is highly sensitive and can accurately measure PTH in serum samples.

A Preclinical Model for Parkinson's Disease Based on Transcriptional Gene Activation via KEAP1/NRF2 to Develop New Antioxidant Therapies.

Investigations of the effect of antioxidants on idiopathic Parkinson's disease have been unsuccessful because the preclinical models used to propose these clinical studies do not accurately represent the neurodegenerative process of the disease. Treatment with certain exogenous neurotoxins induces massive and extremely rapid degeneration; for example, MPTP causes severe Parkinsonism in just three days, while the degenerative process of idiopathic Parkinson´s disease proceeds over many years. The endogenous neurotoxin aminochrome seems to be a good alternative target since it is formed in the nigrostriatal system neurons where the degenerative process occurs. Aminochrome induces all the mechanisms reported to be involved in the degenerative processes of idiopathic Parkinson's disease. The presence of neuromelanin-containing dopaminergic neurons in the postmortem brain of healthy elderly people suggests that neuromelanin synthesis is a normal and harmless process despite the fact that it requires oxidation of dopamine to three ortho-quinones that are potentially toxic, especially aminochrome. The apparent contradiction that neuromelanin synthesis is harmless, despite its formation via neurotoxic ortho-quinones, can be explained by the protective roles of DT-diaphorase and glutathione transferase GSTM2-2 as well as the neuroprotective role of astrocytes secreting exosomes loaded with GSTM2-2. Increasing the expression of DT-diaphorase and GSTM2-2 may be a therapeutic goal to prevent the degeneration of new neuromelanin-containing dopaminergic neurons. Several phytochemicals that induce DT-diaphorase have been discovered and, therefore, an interesting question is whether these phytochemical KEAP1/NRF2 activators can inhibit or decrease aminochrome-induced neurotoxicity.

Human DT-diaphorase expression in Escherichia coli: optimization, purification and structural stability.

Expression and purification of human DT-diaphorase, also referred to as NAD(P)H quinone oxidoreductase 1 (NQO1; EC. 1.6.99.2), which is a flavoprotein belongs to the family of oxidoreductases are optimized. The DT-diaphorase plays an important role in biosensor design for laboratory analysis and also developing biosensor for measurement of glucose level in blood. The aim of this study was to investigate various parameters regarding the expression of DT-diaphorase in Escherichia coli BL21 (DE3) and thermal stability of DT-diaphorase activity at different temperatures in the presence of sucrose. Expression conditions of DT-diaphorase in E. coli were optimized with an induction time (22.00 hr), induction temperature (18.00 ˚C) and also lactose (5.00 mM) and isopropyl ß-D-1-thiogalactopyranoside (1.00 mM) concentrations as inducers. The Km, Vmax and kcat values for NADH as a substrate were 25.50 µM, 357 µM per min and 446.40 µM mg-1 per min, respectively. Results of our research revealed that different concentrations of sucrose at 40.00 ˚C did not have any significant effect on enzyme structure; while, relatively significant changes, especially in the presence of sucrose (0.75 M) at 50.00 ˚C were observed. The results presented show that sucrose causes DT-diaphorase inactivation rate reduction and relatively little increases in thermal stability and thus, sustains its conformation against thermal unfolding.

The diverse functionality of NQO1 and its roles in redox control.

In this review, we summarize the multiple functions of NQO1, its established roles in redox processes and potential roles in redox control that are currently emerging. NQO1 has attracted interest due to its roles in cell defense and marked inducibility during cellular stress. Exogenous substrates for NQO1 include many xenobiotic quinones. Since NQO1 is highly expressed in many solid tumors, including via upregulation of Nrf2, the design of compounds activated by NQO1 and NQO1-targeted drug delivery have been active areas of research. Endogenous substrates have also been proposed and of relevance to redox stress are ubiquinone and vitamin E quinone, components of the plasma membrane redox system. Established roles for NQO1 include a superoxide reductase activity, NAD+ generation, interaction with proteins and their stabilization against proteasomal degradation, binding and regulation of mRNA translation and binding to microtubules including the mitotic spindles. We also summarize potential roles for NQO1 in regulation of glucose and insulin metabolism with relevance to diabetes and the metabolic syndrome, in Alzheimer's disease and in aging. The conformation and molecular interactions of NQO1 can be modulated by changes in the pyridine nucleotide redox balance suggesting that NQO1 may function as a redox-dependent molecular switch.

DT-diaphorase triggered theranostic nanoparticles induce the self-burst of reactive oxygen species for tumor diagnosis and treatment.

On-demand therapy following effective tumor detection would considerably reduce the side effects of traditional chemotherapy. DT-diaphorase (DTD), whose level is strongly elevated in various tumors, is a cytosolic flavoenzyme that promotes intracellular reactive oxygen species (ROS) generation via the redox cycling of hydroquinones. Incorporation of the DTD-responsive substrate to the structures of the probe and prodrug may facilitate the tumor detection and therapy. Herein, we established an multifunctional drug delivery nanosystem (HTLAC) that rapidly responds to the DTD enzyme, leads to the early-stage precise detection and termination of tumors. Firstly, the synthesis of DTD-responsive withaferin A (DT-WA) and indocyanine green (DT-Cy5) was performed. In the presence of DTD, WA, which produces ROS in cells, was released from DT-WA, and the red fluorescence of DT-Cy5 was detected for tumor imaging. Additionally, these DTD enzyme reaction processes of DT-WA and DT-Cy5 induced ROS. The self-burst of ROS generation by the two enzyme reaction processes as well as the released WA then led to the apoptosis of tumor cells. To increase the bioavailability and tumor targeting of drugs, cell-penetrating peptide and hyaluronic acid functionalized liposomes were used to encapsulate the drugs. The detailed in vitro and in vivo assays showed that HTLAC achieved enhanced tumor detection and superior antitumor efficiency. According to above outcomes, results showed that HTLAC might provide an efficacious approach for the fabrication of enzyme-triggering nanosystems to detect tumor and induce the self-burst of ROS for an efficient tumor treatment. STATEMENT OF SIGNIFICANCE: We have fabricated a HTLAC nanosystem to address the need of bursting reactive oxygen species (ROS) generation within tumor site. Our goal uniquely aims at not only augmentation of ROS-inducing anticancer efficacy, but also to meet the challenges of tumor dynamic detection in the clinical practices. In this work, the DT-diaphorase responsive withaferin A (DT-WA) and indocyanine green (DT-Cy5) are synthesized, and observed more specifically toward DTD under physiological conditions. As the cell-penetrating peptide and hyaluronic acid functionalized liposome, the HTLAC not only induces antiproliferative activity by generating self-burst of ROS, but also effectively accumulate and restore its fluorescence at the tumor site because of the HA actively targeting tumor along with the prolonged presence in blood circulation. Besides, this enzyme-triggering nanosystem exhibited an effective tumor inhibition with a low systemic toxicity.

From in vitro to in silico: Modeling and recombinant production of DT-Diaphorase enzyme.

DT-Diaphorase (DTD) belonging to the oxidoreductase family, is among the most important enzymes and is of great significance in present-day biotechnology. Also, it has potential applications in glucose and pyruvate biosensors. Another important role of the DTD enzyme is in the detection of Phenylketonuria disease. According to the above demands, at first, we tried to study molecular cloning and production of recombinant DTD in E. coli BL21 strain. We have successfully cloned, expressed, and purified functionally active diaphorase. The amount of enzyme was increased in 10-h using IPTG induction, and the recombinant protein was purified by Ni-NTA agarose affinity chromatography. After that, the kinetic and thermodynamic parameters of the enzyme, optimum temperature and pH were also investigated to find more in-depth information. In the end, to represent the connections between the structures and function of this enzyme, the molecular dynamics simulations have been considered at two temperatures in which DTD had maximum and minimum activity (310 and 293 K, respectively). The results of MD simulations indicated that the interaction between NADH with phenylalanine 232 residue at 310 K is more severe than other residues. So, to investigate the interaction details of NADH/PHE 232 the DFT calculations were done.

DT-Diaphorase Prevents Aminochrome-Induced Lysosome Dysfunction in SH-SY5Y Cells.

Aminochrome has been reported to induce lysosomal dysfunction by inhibiting the vacuolar H-type ATPase localized in lysosome membrane. DT-diaphorase has been proposed to prevent aminochrome neurotoxicity but it is unknown whether this enzyme prevents aminochrome-induced lysosomal dysfunction. In the present study, we tested the protective role of DT-diaphorase in lysosomal dysfunction by generating a cell line (SH-SY5YsiNQ7) with a stable expression of a siRNA against DT-diaphorase with only 10% expression of mRNA enzyme. The cells differentiated with retinoic acid and 12-o-tetradecanoylphorbol-13-acetate show a significant increase in the expression of tyrosine hydroxylase, vesicular monoamine transporter-2, and dopamine transporter. The incubation of SH-SY5YsiNQ7 cells with 10 µM aminochrome resulted in a significant decrease of lysosome pH determined by using acridine orange, while aminochrome has no effect on SH-SY5Y cells. These results support the proposed protective role of DT-diaphorase against aminochrome-induced lysosomal dysfunction.

Ultrasensitive Detection of Parathyroid Hormone through Fast Silver Deposition Induced by Enzymatic Nitroso Reduction and Redox Cycling.

Enzymatically induced silver deposition and subsequent electrochemical oxidation have been widely used in electrochemical biosensors. However, this method is ineffective for producing highly enhanced silver deposition for use in ultrasensitive detection. Herein, we report a fast silver deposition method that simultaneously uses three signal amplification processes: (i) enzymatic amplification, (ii) chemical-chemical (CC) redox cycling, and (iii) chemical-enzymatic (CN) redox cycling. DT-diaphorase (DT-D) is used for enzymatic amplification to convert a nitroso compound, a species incapable of directly reducing Ag+ to an amine compound, which can directly reduce Ag+. NADH acts as a reducing agent for the indirect reduction of Ag+ via the two redox cycling processes. 4-Nitroso-1-naphthol is converted to 4-amino-1-naphthol (NH2-N) in the presence of DT-D. NH2-N initiates two redox cycling processes: NH2-N, along with Ag+ and NADH, are involved in the CC redox cycling, whereas NH2-N, along with Ag+, DT-D, and NADH, are involved in the CN redox cycling. Finally, the deposited silver is electrochemically oxidized to produce a signal. When this triple signal amplification strategy for fast silver deposition is applied to an electrochemical immunosensor for detecting parathyroid hormone (PTH), a detection limit as low as ∼100 fg/mL is obtained. The concentrations of PTH in clinical serum determined using the developed immunosensor are found to agree with those measured using a commercial instrument. Thus, the use of this strategy for fast silver deposition is highly promising for ultrasensitive electrochemical detection and biosensing applications.

Carboxyl Esterase-Like Activity of DT-Diaphorase and Its Use for Signal Amplification.

Carboxyl esterases show limited use as catalytic labels in bioassays because of slow enzymatic reaction. We report that DT-diaphorase from Bacillus stearothermophilus (DT-D, EC 1.6.99.-) shows high carboxyl esterase-like activity in the presence of reduced ß-nicotinamide adenine dinucleotide (NADH) and may be used as a better catalytic label than carboxyl esterases. DT-D is a redox enzyme and can participate in signal-amplifying redox cycling. Thus, an electrochemical immunosensor using a DT-D label allows for triple signal amplification based on (i) hydrolysis of a carboxyl ester, (ii) electrochemical-chemical (EC) redox cycling involving an electrode, a hydrolysis product, and NADH, and (iii) electrochemical-enzymatic (EN) redox cycling involving an electrode, a hydrolysis product, DT-D, and NADH. Ester hydrolysis by DT-D is confirmed via spectrophotometric measurement of a chromogenic substrate (4-nitrophenyl acetate) and 1H NMR spectra. Among two phenyl acetates and four naphthyl acetates considered, 4-aminonaphthalene-1-yl acetate (4-NH2-NAc) is chosen as the best acetyl ester substrate because 4-NH2-NAc is stable, its hydrolysis is slow in the absence of DT-D, its hydrolysis is very fast in the presence of DT-D, and EC and EN redox cycling involving the hydrolysis product (4-amino-1-naphthol) is rapid. However, hydrolysis of 4-NH2-NAc by esterase from porcine liver (EC 3.1.1.1.) is very slow. When DT-D is applied to sandwich-type detection of thyroid-stimulating hormone in artificial serum, the detection limit is ∼2 pg/mL, indicating that the developed immunosensor is highly sensitive because of triple signal amplification. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common alkaline phosphatase and horseradish peroxidase.

NAD(P)H quinone oxidoreductase (NQO1): an enzyme which needs just enough mobility, in just the right places.

NAD(P)H quinone oxidoreductase 1 (NQO1) catalyses the two electron reduction of quinones and a wide range of other organic compounds. Its physiological role is believed to be partly the reduction of free radical load in cells and the detoxification of xenobiotics. It also has non-enzymatic functions stabilising a number of cellular regulators including p53. Functionally, NQO1 is a homodimer with two active sites formed from residues from both polypeptide chains. Catalysis proceeds via a substituted enzyme mechanism involving a tightly bound FAD cofactor. Dicoumarol and some structurally related compounds act as competitive inhibitors of NQO1. There is some evidence for negative cooperativity in quinine oxidoreductases which is most likely to be mediated at least in part by alterations to the mobility of the protein. Human NQO1 is implicated in cancer. It is often over-expressed in cancer cells and as such is considered as a possible drug target. Interestingly, a common polymorphic form of human NQO1, p.P187S, is associated with an increased risk of several forms of cancer. This variant has much lower activity than the wild-type, primarily due to its substantially reduced affinity for FAD which results from lower stability. This lower stability results from inappropriate mobility of key parts of the protein. Thus, NQO1 relies on correct mobility for normal function, but inappropriate mobility results in dysfunction and may cause disease.

Cancer-associated variants of human NQO1: impacts on inhibitor binding and cooperativity.

Human NAD(P)H quinone oxidoreductase (DT-diaphorase, NQO1) exhibits negative cooperativity towards its potent inhibitor, dicoumarol. Here, we addressed the hypothesis that the effects of the two cancer-associated polymorphisms (p.R139W and p.P187S) may be partly mediated by their effects on inhibitor binding and negative cooperativity. Dicoumarol stabilized both variants and bound with much higher affinity for p.R139W than p.P187S. Both variants exhibited negative cooperativity towards dicoumarol; in both cases, the Hill coefficient (h) was approximately 0.5 and similar to that observed with the wild-type protein. NQO1 was also inhibited by resveratrol and by nicotinamide. Inhibition of NQO1 by resveratrol was approximately 10,000-fold less strong than that observed with the structurally similar enzyme, NRH quinine oxidoreductase 2 (NQO2). The enzyme exhibited non-cooperative behaviour towards nicotinamide, whereas resveratrol induced modest negative cooperativity (h = 0.85). Nicotinamide stabilized wild-type NQO1 and p.R139W towards thermal denaturation but had no detectable effect on p.P187S. Resveratrol destabilized the wild-type enzyme and both cancer-associated variants. Our data suggest that neither polymorphism exerts its effect by changing the enzyme's ability to exhibit negative cooperativity towards inhibitors. However, it does demonstrate that resveratrol can inhibit NQO1 in addition to this compound's well-documented effects on NQO2. The implications of these findings for molecular pathology are discussed.

On the Role of DT-Diaphorase Inhibition in Aminochrome-Induced Neurotoxicity In Vivo.

Dopamine oxidation in the pathway leading to neuromelanin formation generates the ortho-quinone aminochrome, which is potentially neurotoxic but normally rapidly converted by DT-diaphorase to nontoxic leukoaminochrome. However, when administered exogenously into rat striatum, aminochrome is able to produce damage to dopaminergic neurons. Because of a recent report that substantia nigra pars compacta (SNpc) tyrosine hydroxylase (T-OH) levels were unaltered by aminochrome when there was cell shrinkage of dopaminergic neurons along with a reduction in striatal dopamine release, the following study was conducted to more accurately determine the role of DT-diaphorase in aminochrome neurotoxicity. In this study, a low dose of aminochrome (0.8 nmol) with or without the DT-diaphorase inhibitor dicoumarol (0.2 nmol) was injected into the left striatum of rats. Intrastriatal 6-hydroxydopamine (6-OHDA, 32 nmol) was used as a positive neurotoxin control in other rats. Two weeks later, there was significant loss in numbers of T-OH immunoreactive fibers in SNpc, also a loss in cell density in SNpc, and prominent apomorphine (0.5 mg/kg sc)-induced contralateral rotations in rats that had been treated with aminochrome, with aminochrome/dicoumarol, or with 6-OHDA. Findings demonstrate that neurotoxic aminochrome is able to exert neurotoxicity only when DT-diaphorase is suppressed-implying that DT-diaphorase is vital in normally suppressing toxicity of in vivo aminochrome, generated in the pathway towards neuromelanin formation.

Computational Optimization of Bioanalytical Parameters for the Evaluation of the Toxicity of the Phytomarker 1,4 Napthoquinone and its Metabolite 1,2,4-trihydroxynapththalene.

OBJECTIVES: Lawsone (1,4 naphthoquinone) is a non redox cycling compound that can be catalyzed by DT diaphorase (DTD) into 1,2,4-trihydroxynaphthalene (THN), which can generate reactive oxygen species by auto oxidation. The purpose of this study was to evaluate the toxicity of the phytomarker 1,4 naphthoquinone and its metabolite THN by using the molecular docking program AutoDock 4. METHODS: The 3D structure of ligands such as hydrogen peroxide (H2O2), nitric oxide synthase (NOS), catalase (CAT), glutathione (GSH), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH) and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) were drawn using hyperchem drawing tools and minimizing the energy of all pdb files with the help of hyperchem by MM(+) followed by a semi-empirical (PM3) method. The docking process was studied with ligand molecules to identify suitable dockings at protein binding sites through annealing and genetic simulation algorithms. The program auto dock tools (ADT) was released as an extension suite to the python molecular viewer used to prepare proteins and ligands. Grids centered on active sites were obtained with spacings of 54 × 55 × 56, and a grid spacing of 0.503 was calculated. Comparisons of Global and Local Search Methods in Drug Docking were adopted to determine parameters; a maximum number of 250,000 energy evaluations, a maximum number of generations of 27,000, and mutation and crossover rates of 0.02 and 0.8 were used. The number of docking runs was set to 10. RESULTS: Lawsone and THN can be considered to efficiently bind with NOS, CAT, GSH, GR, G6PDH and NADPH, which has been confirmed through hydrogen bond affinity with the respective amino acids. CONCLUSION: Naphthoquinone derivatives of lawsone, which can be metabolized into THN by a catalyst DTD, were examined. Lawsone and THN were found to be identically potent molecules for their affinities for selected proteins.