Selective accumulation of a novel antimalarial rhodacyanine derivative, SSJ-127, in an organelle of Plasmodium berghei

SSJ-127, a novel antimalarial rhodacyanine derivative, has shown potent antimalarial activity against chloroquine-resistant Plasmodium strains in vitro and subcutaneous administration of SSJ-127 results in a complete cure of a mouse malaria model. SSJ-127 was detected by fluorescence microscopy in the mouse malaria parasites Plasmodium berghei after exposure of infected red blood cells to the compound in vitro and in vivo. Selective accumulation of SSJ-127 in an organelle is observed in all blood stages of live malaria parasites. The organelle is clearly different from the mitochondrion and the nucleus in terms of morphology. The shape of the organelle changed during the asexual blood stages of the parasite. There was always a close association between the organelle and the mitochondrion. These results raised the possibility that SSJ-127 accumulates in an apicoplast of the malaria parasite and affects protozoan parasite-specific pathways.     

Purification and characterization of cold-active L-glutamate dehydrogenase independent of NAD(P) and oxygen

L-Glutamate dehydrogenase (GLDH) independent of NAD(P) and oxygen was first obtained from the psychrotrophic bacterium Aeromonas sp. L101, originally isolated from the organs of salmon (Oncorhynchus keta). GLDH was purified by a series of chromatography steps on DEAE-Sepharose, Superdex 200pg, Q-Sepharose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determined to have a molecular mass of 110 kDa and a pI of 5.7. Maximum activity was obtained at 55 degrees C and pH 8.5. The activity of GLDH at 4 and 20 degrees C was 38 and 50%, respectively, of that at 50 degrees C. GLDH was coupled to cytochrome c and several redox dyes including 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS), 2, 6-dichlorophenylindophenol (DCIP), 9-dimethylaminobenzo[alpha]phenoxazin-7-ium chloride (meldola's blue), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4, 4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H tetrazolium chloride] (nitroblue tetrazolium; NBT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H tetrazolium (INT). The presence of NAD(P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profile and ICP data indicated a b-type cytochrome containing iron.     

Plasmodium berghei proteome changes in response to SSJ-183 treatment

The benzo[a]phenoxazine derivative, SSJ-183 has shown excellent anti-malarial efficacy and safety. However, its mechanism of action is unclear. We investigated the effect of SSJ-183 on the rodent malarial parasite, Plasmodium berghei. We analyzed changes in protein expression in the erythrocytic cycle of P. berghei with or without 18 h of SSJ-183 treatment by two-dimensional gel electrophoresis. We confirmed results with matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry. After treatment, seven main proteins were significantly down-regulated, and two were up-regulated; results were reproduced in three independent tests. Some of these proteins were hypothetical parasite proteins or unnamed host products. However, three proteins were identified as a heat shock protein, a disulfide isomerase precursor, and berghepain-2 from P. berghei. All three showed reduced expression after SSJ-183 treatment. This suggested that SSJ-183 was a good anti-malarial drug candidate because it targeted parasite chaperone proteins.     

Inhibitors of nitrofuran reduction in Escherichia coli: evidence for their existence, partial purification, binding of nitrofurantoin in vitro, and implications for nitrofuran resistance

Nitrofurantoin (NF)-resistant mutants of Escherichia coli were isolated as described previously (18). One of the mutants (SSJ-2) was found to possess NF reductase activity equal to that of its parent (E. coli KL16). Two NF-resistant transductional derivatives, SSJ-2A and SSJ-2B, were isolated using SSJ-2 as the donor. SSJ-2 was found to be a double mutant carrying two mutations, nfnA and nfnB, while SSJ-2A (nfnA) and SSJ-2B (nfnB) carried these mutations individually. Heated extracts from SSJ-2A and SSJ-2B were found to inhibit the reduction of NF by unheated extracts of the NF-sensitive strain E. coli KL16 in vitro. Unheated extracts of these mutants reduced NF poorly relative to E. coli KL16. The poor reduction of NF by unheated extracts of SSJ-2A and SSJ-2B was greatly stimulated by heated extracts of SSJ-2B and SSJ-2A, respectively, and also by heated extracts of E. coli KL16. When heated extracts of SSJ-2A and SSJ-2B were mixed in a particular ratio and added to unheated extracts of E. coli KL16 they lost their inhibitory activity. Two proteins, designated inhibitor A and inhibitor B, have been partially purified from heated extracts of SSJ-2B and SSJ-2A, respectively. Their respective molecular weights, as determined by gel chromatography, were 37,000 and 20,500. The two inhibitors bound nitrofurantoin in vitro, and the NF-binding ability was lost when mixed in the molar ration of 3/1 (B/A). These observations were rationalized in terms of a hypothesis which explains (i) maximal NF reduction in wild-type cells, (ii) maximal NF reduction of nfnA-nfnB- double mutant, and (iii) poor NF reduction in nfnA- or nfnB- single mutants. The possible role of these inhibitors in nitrofurantoin resistance is also discussed.     

Potentiation of antimalarial drug action by chlorpheniramine against multidrug-resistant Plasmodium falciparum in vitro

Chlorpheniramine, a histamine H1 receptor antagonist, was assayed for in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum K1 strain and chloroquine-resistant P. falciparum T9/94 clone, by measuring the 3H-hypoxanthine incorporation. Chlorphenirame inhibited P. falciparum K1 and T9/94 growth with IC50 values of 136.0+/-40.2 microM and 102.0+/-22.6 microM respectively. A combination of antimalarial drug and chlorpheniramine was tested against resistant P. falciparum in vitro. Isobologram analysis showed that chlorpheniramine exerts marked synergistic action on chloroquine against P. falciparum K1 and T9/94. Chlorpheniramine also potentiated antimalarial action of mefloquine, quinine or pyronaridine against both of the resistant strains of P. falciparum. However, chlorpheniramine antagonism with artesunate was obtained in both P. falciparum K1 and T9/94. The results in this study indicate that antihistaminic drugs may be promising candidates for potentiating antimalarial drug action against drug resistant malarial parasites.     

Antimalarial chemotherapy: young guns or back to the future?

The burgeoning global problem of malaria is largely due to the emergence of parasite resistance to our limited armamentarium of antimalarial drugs. The recognition of this impending disaster at the international level and the engagement of the pharmaceutical industry promise a more optimistic future for antimalarial drug development. This is particularly exciting when considering the advances in our understanding of parasite biology, which are currently being fuelled by the malaria genome project. This article discusses recent developments in the area of antimalarial drug discovery and evaluation. New advances, based on traditional antimalarial drug classes including the quinolines, peroxides and antifolates (‘back to the future’), are discussed, followed by a presentation of some novel targets (‘young guns’) that have been shown to be good candidates for chemotherapeutic attack.     

Novel synthetic route for antimalarial benzo[a]phenoxazine derivative SSJ-183 and two active metabolites

A productive synthesis of benzo[a]phenoxazine derivative SSJ-183 (1), a promising lead for antimalarial agents, was developed using a one pot procedure. Furthermore, N-deethylated metabolite 3 and bis-N,N-deethylated metabolite 4 were synthesized by the application of the method. The metabolites 3 and 4 showed comparable and ∼2-fold increased activities against drug-sensitive and drug-resistant Plasmodium falciparum parasites.     

Synthesis and in vitro antiprotozoal activities of 5-phenyliminobenzo[a]phenoxazine derivatives

A series of 5-phenyliminobenzo[a]phenoxazine derivatives were synthesized. The in vitro antiprotozoal activities were evaluated against Plasmodium falciparum K1, Trypanosoma cruzi, Leishmania donovani and Trypanosoma brucei rhodesiense. N,N-Diethyl-5-((4-methoxyphenyl)imino)-5H-benzo[a]phenoxazin-9-amine shows IC(50)=0.040 μmol L(-1) with a selective index of 1425 against Plasmodium falciparum K1.     

Antimalarial chemotherapeutic peroxides: artemisinin,yingzhaosu A and related compounds

Mechanism-based rational design and gram-scale chemical synthesis have produced some new trioxane and endoperoxide antimalarial drug candidates that are efficacious and safe. This review summarises recent achievements in this area of peroxide drug development for malaria chemotherapy.     

Prodrug thiamine analogs as inhibitors of the enzyme transketolase

Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.     

Synthesis, antimalarial, antileishmanial, antimicrobial, cytotoxicity, and methemoglobin (MetHB) formation activities of new 8-quinolinamines

We report the synthesis, in vitro antiprotozoal (against Plasmodium and Leishmania), antimicrobial, cytotoxicity (Vero and MetHb-producing properties), and in vivo antimalarial activities of two series of 8-quinolinamines. N1-{4-[2-(tert-Butyl)-6-methoxy-8-quinolylamino]pentyl}-(2S/2R)-2-aminosubstitutedamides (21-33) and N1-[4-(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2S/2R)-2-aminosubstitutedamides (51-63) were synthesized in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5-pentyloxyaniline, respectively. Several analogs displayed promising antimalarial activity in vitro against Plasmodium falciparum D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) clones with high selectivity indices versus mammalian cells. The most promising analogs (21-24) also displayed potent antimalarial activity in vivo in a Plasmodium berghei-infected mouse model. Most interestingly, many analogs exhibited promising in vitro antileishmanial activity against Leishmania donovani promastigotes, and antimicrobial activities against a panel of pathogenic bacteria and fungi. Several analogs, notably 21-24, 26-32, and 60, showed less MetHb formation compared to primaquine indicating the potential of these compounds in 8-quinolinamine-based antimalarial drug development.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

8-Quinolinamines conjugated with amino acids are exhibiting potent blood-schizontocidal antimalarial activities

Alanine, lysine, ornithine and valine conjugated to primaquine and other 8-quinolinamine antimalarials were prepared for blood-schizontocidal antimalarial activity evaluation. The analogues were examined in vivo against Plasmodium berghei (drug-sensitive strain) and Plasmodium yoelii nigeriensis (highly virulent multi-drug-resistant strain) infected mice models. N(1)-[4-(5-Butoxy-4-ethyl-6-methoxy-8-quinolylamino)pentyl]-(2S)-2,6-diaminohexanamide (20) which showed curative activity at 5mg/kg in the P. berghei test emerged as the most effective compound. N(1)-[4-(4-Ethyl-5-hexoxy-6-methoxy-8-quinolylamino)pentyl]-(2S)-2,6-diaminohexanamide (22) exhibited curative activity at 50mg/kg against P. yoelii nigeriensis in mice and emerged as the most potent analogue against multi-drug resistant strain. The results of this study represent development of highly potent 8-quinolinamines for antimalarial drug development.     

Reactivation of Cyclosarin-inhibited Rat Brain Acetylcholinesterase by Pyridinium–Oximes

Cyclohexyl methylphosphonofluoridate (cyclosarin, cyclosin, GF) is a highly toxic organophosphate, which is resistant to conventional oxime therapy. To gain insight into the reactivation kinetics, rat brain acetylcholinesterase (AChE) was inhibited in vitro by cyclosarin (pH 8.0, 25°C) and reactivated with 22 different pyridinium–oximes. Three compounds were shown to be superior to the other oximes: 4-carbamoyl-4′-[(hydroxyimino)methyl]-1,1′-(oxydimethylene)dipyridin-1-ium dichloride (HS-6), 4′-carbamoyl-2-[(hydroxyimino)methyl]-1,1′-(oxydimethylene)dipyridin-1-ium dichloride (HI-6), and 4′-carbamoyl-2-[(hydroxyimino)methyl]-1,1′-(but-2-ene-1,4-diyl)dipyridin-1-ium dichloride (BI-6).     

Design,Synthesis of Novel Lipids as Chemical Permeation Enhancers and Development of Nanoparticle System for Transdermal Drug Delivery

In the present study, we designed and developed novel lipids that include (Z)-1-(Octadec-9-en-1-yl)-pyrrolidine (Cy5T), 1, 1-Di-((Z)-octadec-9-en-1-yl)pyrrolidin-1-ium iodide (Cy5), (Z)-1-(Octadec-9-en-1-yl)-piperidine (Cy6T), and 1, 1-Di-((Z)-octadec-9-en-1-yl) piperidin-1-ium iodide (Cy6) to enhance the transdermal permeation of some selected drugs. Firstly, we evaluated the transdermal permeation efficacies of these lipids as chemical permeation enhancers in vehicle formulations for melatonin, ß-estradiol, caffeine, α-MSH, and spantide using franz diffusion cells. Among them Cy5 lipid was determined to be the most efficient by increasing the transdermal permeation of melatonin, ß-estradiol, caffeine, α-MSH, and spantide by 1.5 to 3.26-fold more at the epidermal layer and 1.3 to 2.5-fold more at the dermal layer, in comparison to either NMP or OA. Hence we developed a nanoparticle system (cy5 lipid ethanol drug nanoparticles) to evaluate any further improvement in the drug penetration. Cy5 lipid formed uniformly sized nanoparticles ranging from 150–200 nm depending on the type of drug. Further, Cy5 based nanoparticle system significantly (p<0.05) increased the permeation of all the drugs in comparison to the lipid solution and standard permeation enhancers. There were about 1.54 to 22-fold more of drug retained in the dermis for the Cy5 based nanoparticles compared to OA/NMP standard enhancers and 3.87 to 66.67-fold more than lipid solution. In addition, epifluorescent microscopic analysis in rhodamine-PE permeation studies confirmed the superior permeation enhancement of LEDs (detection of fluorescence up to skin depth of 340 μm) more than lipid solution, which revealed fluorescence up to skin depth of only 260 μm. In summary the present findings demonstrate that i) cationic lipid with 5 membered amine heterocyclic ring has higher permeating efficacy than the 6 membered amine hertocyclic ring. ii) The nanoparticle system prepared with Cy5 showed significant (p<0.05) increase in the permeation of the drugs than the control penetration enhancers, oleic acid and NMP.     

Reactivation of cyclosarin-inhibited rat brain acetylcholinesterase by pyridinium--oximes

Cyclohexyl methylphosphonofluoridate (cyclosarin, cyclosin, GF) is a highly toxic organophosphate, which is resistant to conventional oxime therapy. To gain insight into the reactivation kinetics, rat brain acetylcholinesterase (AChE) was inhibited in vitro by cyclosarin (pH 8.0, 25 degrees C) and reactivated with 22 different pyridiniumoximes. Three compounds were shown to be superior to the other oximes: 4-carbamoyl-4'-[(hydroxyimino)methyl]-1,1'-(oxydimethylene)dipyridin-1-ium dichloride (HS-6), 4'-carbamoyl-2-[(hydroxyimino)methyl]-1,1'-(oxydimethylene)dipyridin-1-ium dichloride (HI-6), and 4'-carbamoyl-2-[(hydroxyimino)-methyl]-1,1'-(but-2-ene-1,4-diyl)dipyridin-1-ium dichloride (BI-6).     

Synthesis of isoquinuclidine analogs of chloroquine: antimalarial and antileishmanial activity

The isoquinuclidine (2-azabicyclo[2.2.2]octane) ring system may be viewed as a semi-rigid boat form of the piperidine ring and, when properly substituted, a scaffold for rigid analogs of biologically active ethanolamines and propanolamines. It is present in natural products (such as ibogaine and dioscorine) that display interesting pharmacological properties. In this study, we have expanded our continuing efforts to incorporate this ring system in numerous pharmacophores, by designing and synthesizing semirigid analogs of the antimalarial drug chloroquine. The analogs were tested in vitro against Plasmodium falciparum strains and Leishmania donovani promastigote cultures. Compounds 6 and 13 displayed potent antimalarial activity against both chloroquine-susceptible D6 and the -resistant W2 strains of P. falciparum. All analogs also demonstrated significant antileishmanial activity with compounds 6 and 13 again being the most potent. The fact that these compounds are active against both chloroquine-resistant and chloroquine-sensitive strains as well as leishmanial cells makes them promising candidates for drug development.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

Abstract

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Novel series of 1,2,4-trioxane derivatives as antimalarial agents

Among three series of 1,2,4-trioxane derivatives, five compounds showed good in vitro antimalarial activity, three compounds of which exhibited better activity against P. falciparum resistant (RKL9) strain than the sensitive (3D7) one. Two best compounds were one from aryl series and the other from heteroaryl series with IC₅₀ values of 1.24 µM and 1.24 µM and 1.06 µM and 1.17 µM, against sensitive and resistant strains, respectively. Further, trioxane derivatives exhibited good binding affinity for the P. falciparum cysteine protease falcipain 2 receptor (PDB id: 3BPF) with well defined drug-like and pharmacokinetic properties based on Lipinski’s rule of five with additional physicochemical and ADMET parameters. In view of having antimalarial potential, 1,2,4-trioxane derivative(s) reported herein may be useful as novel antimalarial lead(s) in the discovery and development of future antimalarial drug candidates as P. falciparum falcipain 2 inhibitors against resistant malaria.     

A review of antimalarial plants used in traditional medicine in communities in Portuguese-speaking countries: Brazil, Mozambique, Cape Verde, Guinea-Bissau, São Tomé and Príncipe and Angola

The isolation of bioactive compounds from medicinal plants, based on traditional use or ethnomedical data, is a highly promising potential approach for identifying new and effective antimalarial drug candidates. The purpose of this review was to create a compilation of the phytochemical studies on medicinal plants used to treat malaria in traditional medicine from the Community of Portuguese-Speaking Countries (CPSC): Angola, Brazil, Cape Verde, Guinea-Bissau, Mozambique and S?o Tomé and Príncipe. In addition, this review aimed to show that there are several medicinal plants popularly used in these countries for which few scientific studies are available. The primary approach compared the antimalarial activity of native species used in each country with its extracts, fractions and isolated substances. In this context, data shown here could be a tool to help researchers from these regions establish a scientific and technical network on the subject for the CPSC where malaria is a public health problem.