Stanley Mukanganyama

Inhibition of glutathione S-transferases by antimalarial drugs possible implications for circumventing anticancer drug resistance

A strategy to overcome multidrug resistance in cancer cells involves treatment with a combination of the antineoplastic agent and a chemomodulator that inhibits the activity of the resistance‐causing protein. The aim of our study was to investigate the effects of antimalarial drugs on human recombinant glutathione S‐transferase (GSTs) activity in the context of searching for effective and clinically acceptable inhibitors of these enzymes. Human recombinant GSTs heterologously expressed in Escherichia coli were used for inhibition studies. GST A1‐1 activity was inhibited by artemisinin with an IC50 of 6 μM, whilst GST M1‐1 was inhibited by quinidine and its diastereoisomer quinine with IC50s of 12 μM and 17 μM, respectively. GST M3‐3 was inhibited by tetracycline only with an IC50 of 47 μM. GST P1‐1 was the most susceptible enzyme to inhibition by antimalarials with IC50 values of 1, 2, 1, 4, and 13 μM for pyrimethamine, artemisinin, quinidine, quinine and tetracycline, respectively. The IC50 values obtained for artemisinin, quinine, quinidine and tetracycline are below peak plasma concentrations obtained during therapy of malaria with these drugs. It seems likely, therefore, that GSTs may be inhibited in vivo at doses normally used in clinical practice. Using the substrate ethacrynic acid, a diuretic drug also used as a modulator to overcome drug resistance in tumour cells, GST P1‐1 activity was inhibited by tetracycline, quinine, pyrimethamine and quinidine with IC50 values of 18, 27, 45 and 70 μM, respectively. The ubiquitous expression of GSTs in different malignancies suggests that the addition of nontoxic reversing agents such as antimalarials could enhance the efficacy of a variety of alkylating agents.

Lysosomal trapping of amodiaquine: impact on transport across intestinal epithelia models.

The lipophilic weak base amodiaquine is an antimalarial drug that has been in use for over 40 years. Little is known of amodiaquines mechanism of transport across membranes. Transport experiments of amodiaquine in Caco‐2 cells showed a low recovery of 30% and rapid disappearance from the apical chamber. Compounds structurally similar to amodiaquine, and those affecting non‐specific binding of amodiaquine or the pH of the system, were tested to unravel the mechanism behind these observations. Chloroquine and ammonium chloride increased the transmonolayer permeability of amodiaquine and decreased its accumulation in Caco‐2 cells, whereas BSA had no effect. Chloroquine and BSA decreased plastic binding whereas ammonium chloride had no effect. This suggests that amodiaquine is trapped in acidic cell compartments such as lysosomes. Amodiaquine was also trapped in rat intestinal tissue. In addition, permeability from the apical to basolateral direction was significantly higher, suggesting an active uptake over the apical membrane of the rat tissue. It can be concluded that amodiaquine is trapped in acidic cell compartments due to its base properties and recovery may be improved by the use of ammonium chloride rather than BSA in transport experiments. Further studies are required to confirm whether amodiaquine is actively absorbed in the intestine. Copyright

Inhibition of human glutathione transferases by multidrug resistance chemomodulators in vitro

Reversal of the drug-resistance phenotype in cancer cells usually involves the use of a chemomodulator that inhibits the function of a resistance-related protein. The aim of this study was to investigate the effects of MDR chemomodulators on human recombinant glutathione S-transferase (GSTs) activity. IC50 values for 15 MDR chemomodulators were determined using 1-chloro-dinitrobenzene (CDNB), cumene hydroproxide (CuOOH) and anticancer drugs as substrates. GSTs A1, P1 and M1 were inhibited by O6-benzylguanine (IC50s around 30 μM), GST P1-1 by sulphinpyrazone (IC50 = 66 μM), GST A1-1 by sulphasalazine, and camptothecin (34 and 74 μM respectively), and GST M1-1 by sulphasalazine, camptothecin and indomethacin (0.3, 29 and 30 μM respectively) using CDNB as a substrate. When ethacrynic acid (for GST P1-1), CuOOH (for A1-1) and 1,3-bis (2-chloroethyl)-1-nitrosourea (for GST M1-1) were used as substrates, these compounds did not significantly inhibit the GST isoforms. However, progesterone was a potent inhibitor of GST P1-1 (IC50 = 1.4 μM) with ethacrynic acid as substrate. These results suggest that the target of chemomodulators in vivo could be a specific resistance-related protein.

Proposed Reductive Metabolism of Artemisinin by Glutathione Transferases in vitro

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.

Evaluation of Nitrite Radical Scavenging Properties of Selected Zimbabwean Plant Extracts and Their Phytoconstituents

Oxidative stress resulting from accumulation of reactive oxygen species has been associated with disease. The search for natural antioxidants of plant origin is necessitated by the side effects associated with synthetic antioxidants currently available. The objective of the study was to determine the antioxidant activity of Combretum zeyheri, Combretum platypetalum, and Parinari curatellifolia extracts by determining nitrite radical scavenging ability. The nitrite radical scavenging assay was used to evaluate the antioxidant potential of the extracts. The total flavonoid content of P. curatellifolia methanol extract was determined by the aluminium calorimetric method. The aqueous and ethanolic leaf extracts of C. zeyheri, C. platypetalum, and P. curatellifolia extracts exhibited nitrite radical scavenging activity. The results show the scavenging activity in the order of potency: P. curatellifolia > C. platypetalum > C. zeyheri with concentration values of 103 µg/mL, 158 µg/mL, and 188 µg/mL for the ethanol extracts and 92.5 µg/mL, 97.5 µg/mL, and 198 µg/mL for the water extracts, respectively. P. curatellifolia ethanol extract was the most potent and the total flavonoid content was estimated to be 0.4 ± 0.05 mg/g quercetin and could account for the activity. Thus, our findings provide evidence that C. zeyheri, C. platypetalum, and P. curatellifolia leaf extracts could be potential sources of natural antioxidants.

Glutathione transferase from Plasmodium falciparum - Interaction with malagashanine and selected plant natural products

A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC50 values of 8.5, 12, 50 and 69 μM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The Ki for GSH and CDNB were −0.015 μM and 0.011 μM, respectively. Malagashanine (100 µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t1/2 of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5 mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds.

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1.

Glutathione transferase P1-1 is over expressed in some cancer cells and contributes to detoxification of anticancer drugs, leading to drug-resistant tumors. The inhibition of human recombinant GSTP1-1 by natural plant products was investigated using 10 compounds isolated from plants indigenous to Southern and Central Africa. Monochlorobimane and 1-chloro-2,4-dinitrobenzene were used to determine GST activity. Each test compound was screened at 33 and 100 µM. Isofuranonapthoquinone (1) (from Bulbine frutescens) showed 68% inhibition at 33 µM, and sesquiterpene lactone (2) (from Dicoma anomala) showed 75% inhibition at 33 μM. The IC50 value of 1 was 6.8 μM. The mode of inhibition was mixed, partial (G site) and noncompetitive (H site) with Ki values of 8.8 and 0.21 µM, respectively. Sesquiterpene 2 did not inhibit the CDNB reaction. Therefore, isofuranonapthoquinone 1 needs further investigations in vivo because of its potent inhibition of GSTP1-1 in vitro.

Phenotyping of the glutathione S-transferase M1 polymorphism in Zimbabweans and the effects of chloroquine on blood glutathione S-transferases M1 and A.

The frequency of the null allele phenotype of glutathione S-transferase (GST) M1 was investigated in 114 Zimbabweans and results for a subset of 63 subjects were compared with genotyping by PCR. In addition, the effect of the antimalarial chloroquine on blood levels of GSTM1 and GSTA in 19 subjects was studied. Quantification of GSTs was by enzyme linked immunosorbent assays (ELISA). Thirty percent of the subjects were of the GSTM1 null phenotype. Comparison of results of phenotyping by ELISA and genotyping by PCR showed that 16% of samples were in discordance; unknown mutations in the GSTM1 gene in the Zimbabwean population may explain this observation. Chloroquine decreased levels of blood GSTM1 and GSTA by 50% or more. In populations treated with chloroquine, these decreases in GST activities might lead to compromised ability to detoxify xenobiotics, could confound GSTM1 phenotyping and might invalidate use of GSTA as an indicator of liver damage.

Inhibitory Activity of Combretum zeyheri and its S9 Metabolites againstEscherichia coli, Bacillus subtilis and Candida albicans

Medicinal plants are therapeutic resources used by traditional population for health care, thus, it is important to evaluate the mechanism of action of herbal plants so as to validate their efficacy and avoid possible toxic effects. The water and methanol leaf extracts of Combretum zeyheri, medicinal plant from Zimbabwe were screened for antibacterial activity against Escherichia Coli and Bacillus subtilis using the agar disc diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the plant extracts were determined using the broth dilution method. Drug transport assays were conducted to test the potential of plant extracts as inhibitors of drug efflux using in Rhodamine 6G. The effect of Combretum zeyheri plant S9 metabolites on growth of Candida albicans was investigated using S9 fractions from 8 week old male Sprague-Dawley rats. Results showed that the plant extract had antibacterial activity against E. coli and B. subtilis, with the aqueous extract exhibiting a greater inhibition of growth than the methanol extract. The plant extract also showed inhibitory effects on drug efflux in all the three organisms suggesting that the antimicrobial actions could partly be due to inhibition of drug efflux pumps. The S9 metabolites of C. zeyheri also had an inhibitory effect on growth of C. albicans, suggesting they may be inhibitors of the growth of Candida albicans in vivo. Combretum zeyheri, therefore, has significant antimicrobial activity and, therefore, has a basis for its use in traditional herbal medications for the treatment of a broad range of microbial infections. The antifungal effects may be mediated via both parent extracts and their metabolites.

Antifungal Activities and Preliminary Phytochemical Investigation of Combretum Species from Zimbabwe

Aeromonas hydrophila is one of the serious pathogens in ornamental fishes causing hemorrhagic bacterial septicemia leading to inflammation and necrosis of the gastrointestinal tract, kidney, muscle, and spleen. Recent studies demonstrated that immunoproteomic vaccines provide protection against bacterial pathogens in fin fish aquaculture and the vaccines are popular due to its long lasting immunity, safety and low cost versatile characteristics. In this study we used an Outer Membrane Protein (OMP) of Aeromonas hydrophila as a vaccine to provide protection against the pathogen in goldfish (Carassius auratus). We used the extract of Asparagus racemosus as an adjuvant in the vaccine preparation. Surivival and immunological response of the vaccinated fishes (30 and 60 days post vaccination (dpv)), were evaluated after challenge with virulent A. hydrophila. The vaccine treated experimental groups significantly improved (P<0.05) the survival at 50% compared to the controls and had improved immunological responses including phagocytosis, albumin-globulin ratio, serum bactericidal activity, and serum lysozyme activity.