Chloroquine-resistant Parasites Research Articles

Persistence of chloroquine-resistant haplotypes of Plasmodium falciparum in children with uncomplicated Malaria in Lagos, Nigeria, four years after change of chloroquine as first-line antimalarial medicine.

BackgroundIn Nigeria, despite the change in National malaria drug policy to artemisinin combination therapy (ACT) in 2005 due to widespread chloroquine resistance, chloroquine (CQ) is still widely used in the treatment of malaria because it is cheap, affordable and accessible. The use of ACT for the management of uncomplicated malaria is currently being promoted. The employment of genetic markers to track circulating chloroquine-resistant parasites are useful in elucidating likely poor efficacy of chloroquine, especially in settings where it is not recommended for the treatment of uncomplicated falciparum malaria. This study determined the prevalence of pfcrt haplotypes and point mutations in pfmdr1 genes four years after the change in antimalarial treatment policy from CQ to the ACTs in Lagos, a commercial city in South-West, Nigeria.MethodsThis was a cross sectional study on uncomplicated malaria in children less than 12 years that presented with fever and other symptoms suggestive of malaria. Parasite DNA was extracted from 119 patients out of 251 children who were positive for Plasmodium falciparum by microscopy and amplified. The occurrence of haplotypes was investigated in pfcrt gene using probe-based qPCR and single nucleotide polymorphisms in pfmdr1 gene using nested PCR.ResultsOne hundred and nine (109) of the 119 children with P falciparum infection (91.6%) harbourd parasites with the mutant pfcrt haplotype (CVIET). Out of this, 4.2% comprised a mixture of genotypes encoding CVMNK and CVIET, while 4.2% had the wild type (CVMNK). Furthermore, the frequency of point mutations in pfmdr1 was 62.2% and 69.0% for codons Y86 and F184 respectively. There were no mutations at codons 1034, 1042 and 1246 of the Pfmdr1 genes.ConclusionThe high frequency of the CQ-resistant haplotypes (CVIET) and mutations in Pfmdr1 associated with CQ resistance in P. falciparum among these children suggest that CQ-resistant parasites are still in circulation. Continuous use of chloroquine may continue to increase the level of mutations in pfcrt and pfmdr1genes. There is need to strengthen current case management efforts at promoting ACT use as well as urgently restricting access to chloroquine by the National drug regulatory agency, National Agency for Food Drug Administration and Control (NAFDAC).Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2069472010142303

Synthesis and evaluation of the antiplasmodial activity of tryptanthrin derivatives

Malaria remains one of the most deadly diseases threatening humankind and is still affecting a significant proportion of the world population, especially in Africa. Chemotherapy is a vital component of the fight against the disease and new antimalarial agents are urgently needed to curb the spread of malaria parasites that are resistant to existing drugs. The natural product tryptanthrin is known for its wide range of activities, including antiplasmodial activity, but its poor solubility has undermined its development as potent antimicrobial and antiprotozoan agent. The aim of this work was to synthesize analogues of tryptanthrin and to evaluate their antiplasmodial activity against the asexual and sexual blood stages of Plasmodium falciparum. Our results suggest that most tryptanthrin analogues retained their antiplasmodial activity against chloroquine-sensitive and chloroquine-resistant malaria parasites in the nanomolar range (30–100 nM). The antiplasmodial activity of the most active compound NT1 (IC50: 30 nM; SI: 155.9) was similar in both strains and close to that of chloroquine (IC50: 20 nM) on the sensitive strain. The antiplasmodial activity was improved with derivatization, thus pointing out the necessity to explore tryptanthrin using medicinal chemistry approaches. Ten (10) of the tested derivatives met the criteria, allowing for advancement to animal testing, i.e., SI > 100 and IC50 < 100 nM. In addition to their activity on the asexual stages, tryptanthrin and two selected derivatives (NT1 and T8) prevented the maturation of gametocytes at their IC90 concentrations, indicating a transmission-blocking potential. Moreover, NT1 was able to impair gametogenesis by reducing the exflagellation of microgametes by 20% at IC90, while tryptanthrin and T8 had no influence on exflagellation. The results of this study confirm that tryptanthrin and its derivatives are potential antimalarial candidates with abilities to kill the intraerythrocytic asexual stages and prevent the formation of sexual stages of the parasite.

Assessing anti-malarial drug effects ex vivo using the haemozoin detection assay.

BackgroundIn vitro sensitivity assays are crucial to detect and monitor drug resistance. Plasmodium falciparum has developed resistance to almost all anti-malarial drugs. Although different in vitro drug assays are available, some of their inherent characteristics limit their application, especially in the field. A recently developed approach based on the flow cytometric detection of haemozoin (Hz) allowed reagent-free monitoring of parasite maturation and detection of drug effects in culture-adapted parasites. In this study, the set-up, performance and usefulness of this novel assay were investigated under field conditions in Gabon.MethodsAn existing flow cytometer (Cyflow Blue) was modified on site to detect light depolarization caused by Hz. Blood from malaria patients was incubated for 72 hrs with increasing concentrations of chloroquine, artesunate and artemisinin. The percentage of depolarizing red blood cells (RBC) was used as maturation indicator and measured at 24, 48 and 72 hrs of incubation to determine parasite growth and drug effects.ResultsThe flow cytometer was easily adapted on site to detect light depolarization caused by Hz. Analysis of ex vivo cultures of parasites, obtained from blood samples of malaria patients, showed four different growth profiles. In 39/46 samples, 50% inhibitory concentrations (IC50) were successfully determined. IC50 values for chloroquine were higher than 200 nM in 70% of the samples, indicating the presence of chloroquine-resistant parasites. For artesunate and artemisinin, IC50 values ranged from 0.9 to 60 nM and from 2.2 nM to 124 nM, respectively, indicating fully sensitive parasites.ConclusionFlow cytometric detection of Hz allowed the detection of drug effects in blood samples from malaria patients, without using additional reagents or complex protocols. Adjustment of the initial parasitaemia was not required, which greatly simplifies the protocol, although it may lead to different IC50 values. Further investigation of set-up conditions of the Hz assay, as well as future studies in various settings should be performed to further determine the usefulness of this assay as a tool for rapid resistance testing in malaria-endemic countries.

The antimalarial activity of 4-aminoalcohol quinoline enantiomers

Introduction: Plasmodium falciparum is a deadly parasite that has developed resistance to existing drugs. 4-Amino-alcohols (4-AQ) are potent antimalarial drugs (e.g. chloroquine, mefloquine and quinine) that block the process of transforming heam to heamozoin in the digestive vacuole of the parasite. Objectives: Given four derivatives of 4-AQ the question addressed was what aspect of the molecule enhances its antimalarial activity in Dd2 chloroquine-resistant parasites? Materials and methods: To determine the activity of four derivatives of 4-AQ by measuring their ‘ IC50’ (i.e. dose required to inhibit growth of the parasite by 50%) using a SYBR Green I malaria drug sensitivity assay. Results: The five-carbon derivative was found to be more effective than the seven-carbon derivative. Furthermore, (R )-enantiomers were found to be less active than their (S )-enantiomer counterparts. Conclusions: Data from this study provides a hypothetical model for amino alcohol uptake, action and proposed resistance in P. falciparum . Acknowledgements: Dr Paul Horrocks (Keele University) for use of his laboratory, Mr Imran Ullah (Keele University) for supervising and helping us with this project, Catherin Mullie for providing drug samples and Dr Waleed Alshaqha for supporting us in our project trip.

Synthesis of gallinamide A analogues as potent falcipain inhibitors and antimalarials.

Analogues of the natural product gallinamide A were prepared to elucidate novel inhibitors of the falcipain cysteine proteases. Analogues exhibited potent inhibition of falcipain-2 (FP-2) and falcipain-3 (FP-3) and of the development of Plasmodium falciparum in vitro. Several compounds were equipotent to chloroquine as inhibitors of the 3D7 strain of P. falciparum and maintained potent activity against the chloroquine-resistant Dd2 parasite. These compounds serve as promising leads for the development of novel antimalarial agents.

Antiplasmodial activity of chloroquine analogs against chloroquine-resistant parasites, docking studies and mechanisms of drug action.

BackgroundGiven the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated.MethodsSix diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (HssLDH) or plasmodial lactate dehydrogenase (PfLDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro.ResultsAll tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of PfLDH; specificity between the residues involved in H-bonds of the PfLDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of PfLDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive.ConclusionsThe presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than PfLDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.

Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon.

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies.

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.

High prevalence of pfcrt-CVIET haplotype in isolates from asymptomatic and symptomatic patients in south-central Oromia, Ethiopia

BackgroundAs a result of extensive chloroquine resistance (CQR) in Plasmodium falciparum in late 1990s, Ethiopia replaced CQ with sulphadoxine-pyrimethamine (SP) as first-line drug, which in turn was replaced by artemisinin combination therapy in 2004. Plasmodium falciparum resistance to CQ is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene. Understanding diversity in the P. falciparum genome is crucial since it has the potential to influence important phenotypes of the parasite such as drug resistance. Limited data is available regarding the type of pfcrt mutant allelic type, the effect of CQ withdrawal and diversity of the parasite population in south-central Oromia, Ethiopia.MethodsFinger-pricked blood spotted on Whatman 3MM filter papers were collected from falciparum malaria patients. Parasite DNA was extracted from individual blood spots on the filter papers. The presence of K76T mutations was determined using nested PCR for all isolates. Complete sequencing of mutations in pfcrt 72-76 was done for a set of randomly selected resistant isolates. Four microsatellite (MS) markers were analysed to determine the heterozygosity.ResultsAlthough CQ was withdrawn for more than a decade, 100% of the parasites still carried the pfcrt K76T mutation. All isolates were mutant at the K76T polymorphism. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Heterozygosity (He) for MS flanking the pfcrt chloroquine resistance allele ranged from 0.00 (mscrt -29, -29.268 kb) to 0.21 (mscrt -2, -2.814 kb). He ranged from 0.00 (msint 3, 0 kb) to 0.19 (msint 2, 0 kb) for MS within the pfcrt gene. Both intronic and MS flanking the pfcrt gene showed low levels of diversity.Conclusionpfcrt CQR allele seems to be fixed in the study area. Of the different haplotypes associated with CQR, only the CVIET genotype was identified. No reversal to the wild-type has occurred in Ethiopia unlike in many Africa countries where CQR parasites declined after cessation of CQ use. Decreased diversity in CQR isolates surrounding pfcrt suggests CQ selection and homogenization among CQR parasite population. While mutation in msint 3 and mscrt -29 of the mutant pfcrt allele is being fixed, it seems that mutations in msint 2 and mscrt -2 are still evolving and may indicate the start of re-diversification of the population from a fixed 76 T population.

Chlorpheniramine Analogues Reverse Chloroquine Resistance in Plasmodium falciparum by Inhibiting PfCRT.

The emergence and spread of malaria parasites that are resistant to chloroquine (CQ) has been a disaster for world health. The antihistamine chlorpheniramine (CP) partially resensitizes CQ-resistant (CQR) parasites to CQ but possesses little intrinsic antiplasmodial activity. Mutations in the parasite's CQ resistance transporter (PfCRT) confer resistance to CQ by enabling the protein to transport the drug away from its site of action, and it is thought that resistance-reversers such as CP exert their effect by blocking this CQ transport activity. Here, a series of new structural analogues and homologues of CP have been synthesized. We show that these compounds (along with other in vitro CQ resistance-reversers) inhibit the transport of CQ via a resistance-conferring form of PfCRT expressed in Xenopus laevis oocytes. Furthermore, the level of PfCRT-inhibition was found to correlate well with both the restoration of CQ accumulation and the level of CQ resensitization in CQR parasites.

Metabolic QTL Analysis Links Chloroquine Resistance in Plasmodium falciparum to Impaired Hemoglobin Catabolism

Drug resistant strains of the malaria parasite, Plasmodium falciparum, have rendered chloroquine ineffective throughout much of the world. In parts of Africa and Asia, the coordinated shift from chloroquine to other drugs has resulted in the near disappearance of chloroquine-resistant (CQR) parasites from the population. Currently, there is no molecular explanation for this phenomenon. Herein, we employ metabolic quantitative trait locus mapping (mQTL) to analyze progeny from a genetic cross between chloroquine-susceptible (CQS) and CQR parasites. We identify a family of hemoglobin-derived peptides that are elevated in CQR parasites and show that peptide accumulation, drug resistance, and reduced parasite fitness are all linked in vitro to CQR alleles of the P. falciparum chloroquine resistance transporter (pfcrt). These findings suggest that CQR parasites are less fit because mutations in pfcrt interfere with hemoglobin digestion by the parasite. Moreover, our findings may provide a molecular explanation for the reemergence of CQS parasites in wild populations.

Anti-Plasmodial Polyvalent Interactions in Artemisia annua L. Aqueous Extract – Possible Synergistic and Resistance Mechanisms

Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L-1), dihydroartemisinic acid (70.0±0.3 mg L-1), arteannuin B (1.3±0.0 mg L-1), isovitexin (105.0±7.2 mg L-1) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 μM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B’s potentiation of artemisinin.

Occurrence of multiple chloroquine-resistant Pfcrt haplotypes and emergence of the S(agt)VMNT type in Cameroonian Plasmodium falciparum

The main objective of this study was to unravel the distribution of different Pfcrt genotypes in the central, littoral, eastern and southern regions of Cameroon and also in locations bordering Gabon and Equatorial Guinea. This is because (i) the chloroquine-resistant malaria parasite Plasmodium falciparum shows a wide occurrence in Cameroon, (ii) mutations in the 72nd to 76th amino acid positions of the Pfcrt gene are known to confer resistance to chloroquine, and (iii) only a single chloroquine-resistant haplotype (C72V73I74E75T76) has so far been reported in Cameroon. We followed a molecular approach with DNA sequencing of the second exon of the Pfcrt gene to identify single nucleotide polymorphisms in 180 P. falciparum field isolates sampled in five different locations in Cameroon. The chloroquine-resistant Pfcrt CVIET haplotype was most abundant, followed by the wild-type CVMNK haplotype. Five hitherto unreported chloroquine-resistant Pfcrt haplotypes were detected for the first time in Cameroonian P. falciparum, including the surprise appearance of the S(agt)VMNT haplotype. The high observed haplotype diversity of the chloroquine-resistant Pfcrt gene and the appearance of the S(agt)VMNT haplotype are daunting and can be attributed to drug pressure and/or the misuse of chloroquine and/or amodiaquine in Cameroon.

Ligand Based Validated Comparative Chemometric Modeling and Pharmacophore Mapping of Aurone Derivatives as Antimalarial Agents

Chloroquine resistance is nowadays a great problem. Aurone derivatives are effective against chloroquine resistant parasite. Ligand based validated comparative chemometric modeling through 2D-QSAR and kNN-MFA 3DQSAR studies as well as common feature 3D pharmacophore mapping were done on thirtyfive aurone derivatives having antimalarial activity. Statistically significant 2D-QSAR models were generated on unsplitted as well as splitted dataset by MLR and PLS technique. The MLR model of the unsplitted method was validated by two-deep cross validation and 10 fold cross validation for determining the predictive ability. The PLS technique of the unsplitted method was done to compare the significance of these methods. In the splitted method, model was developed on the training set by Y-based ranking method by using the same descriptors and was validated on fifty pairs of the test and the training sets by k-MCA technique. These models generated by using the same descriptors were well validated irrespective of MLR as well as PLS analysis of unsplitted as well as splitted methods and are showing similar results. Therefore, these descriptors and model generated were reliable and robust. The kNN-MFA 3D-QSAR models were generated by three variable selection methods: genetic algorithm, simulated annealing and stepwise regression. The kNN-MFA 3D-QSAR results support the 2D QSAR data and in turn validate the earlier observed SAR results. Common feature 3D-pharmacophore generation was performed on these compounds to validate both 2D and 3D-QSAR studies as well as the earlier observed SAR data. The work highlights the required structural features for the higher antimalarial activity.

Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum

Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite’s acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (FST=0.253, P<0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r=0.49, P=0.003, N=83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation at both neutral and adaptive loci across India.

Histological and biochemical effects of Arteether&lt;sup&gt;tm&lt;/sup&gt; on the liver of wistar rats

Arteether™ is among the recent drugs that are used to combat chloroquine-resistant malarial parasites. This study examined the effects of arteether™ on enzyme biomarkers of the liver, serum protein concentrations, and liver morphology. Twenty (20) adult albino Wistar rats weighing 200 - 250 g were randomly divided into four groups (A, B, C and D) of five animals each, and used in this study. Group A rats were given intramuscular (i. m.) arteether™ (3 mg/kg b. w.) daily for 3 days. Group B rats received i. m. arteether™ (6 mg/kg b. w.) daily for 3 days. Group C rats were given i. m. arteether™ (3 mg/kg b. w.) daily for 3 days. The same dose was repeated at two-weekly intervals for 4 further weeks, while group D rats which received normal saline (0.9 % w/ v, 3 ml/kg b.w.), served as controls. At the end of the experiment, the body weights of the animals were determined and recorded. Serum levels of alanine transaminase (ALT), aspartate transaminase (ASP), alkaline phosphatase (ALP), total protein (TP) and albumin were assayed, and histological studies were performed. Results obtained show no significant difference (P<0.05) in liver enzymes (ALT, ASP, ALP). TP and albumin were significantly reduced in group C rats. Histological studies revealed no cyto-architectural changes. It is concluded that at therapeutic doses, arteether™ is well tolerated in Wistar rats.

Exploring structural requirements of aurone derivatives as antimalarials by validated DFT-based QSAR, HQSAR, and COMFA–COMSIA approach

Chloroquine resistance is nowadays a great problem in malaria. Aurone derivatives were effective against chloroquine resistant parasite. Validated density functional theory (DFT)-based chemometric modeling, hologram QSAR (HQSAR), comparative molecular field analysis (CoMFA), and comparative molecular similarity analysis (CoMSIA) studies were conducted on 35 aurone derivatives having antimalarial activity. 2D-QSAR models were developed on the training sets by Y-based ranking method. This model was validated on 50 pairs of the test and the training sets by k-Means cluster analysis method. HQSAR, CoMFA, and CoMSIA models were validated by standard techniques and each method validates the DFT-based 2D-QSAR study and in turn validates the earlier observed structural activity relationship data as well as each other. DFT-based 2D-QSAR model suggests that the increase of Mulliken charge at C14 and HOMO density located on C11 may be conducive to antimalarial activity. Ethyl group attached to C14 and the increase of the value of chemical potential may be beneficial for antimalarial activity. Methoxy fragment is important for better antimalarial activity by HQSAR study. CoMFA analysis shows a favorable steric green region is located near C14 whereas the unfavorable yellow region is far away from C14. A large blue region located near C14 indicates the positively charged groups are favorable at this position. CoMSIA steric features correlates well with the CoMFA steric features. CoMSIA study suggests the bulky hydrophobic substitution at C14 is necessary for antimalarial activity. These results may be utilized to obtain potential antimalarial molecules.

Effective treatment with a tetrandrine/chloroquine combination for chloroquine-resistant falciparum malaria in Aotus monkeys

BackgroundIn vitro evidence indicates that tetrandrine (TT) can potentiate the action of chloroquine 40-fold against choloquine-resistant Plasmodium falciparum. The key question emanating from that study is “would tetrandine and chloroquine be highly effective in a live Aotus monkey model with chloroquine-resistant parasites”. This study was designed to closely mimic the pharmacological/anti-malarial activity in man.MethodsThe Vietnam Smith/RE strain of P. falciparum, which is chloroquine-resistant was used in this study. Previous experimental procedures were followed. Panamanian owl monkeys (Aotus) were inoculated with 5×106 erythrocytes parasitized with the CQ-resistant strain of P. falciparum. Oral drug treatment was with CQ (20 mg/kg) and/or tetrandrine at 15 mg/Kg, 30 mg/Kg or 60 mg/Kg or 25 mg/Kg depending on experimental conditions.Results and DiscussionParasitaemia was cleared rapidly with CQ and TT while CQ treatment alone was ineffective. Recrudescence of malaria occurred after seven days post-infection. However, four animals were treated orally with TT and CQ parasites were cleared. It is likely that monkeys were cured via a combination of both drug and host immune responses. A single Aotus monkey infected with P. falciparum and untreated with drugs, died. No side effects were observed with these drug treatments.ConclusionsThis combination of chloroquine and tetrandrine forms the basis of a new attack on chloroquine-resistant malaria - one based upon inhibition of the basis of chloroquine resistance, the multiple drug resistance pump. Previous studies demonstrated that the parasite MDR pump was found on parasite membranes using 3H azidopine photoaffinity labelling.Since MDR-based choloroquine resistance is induced by chloroquine, the basis of the action of tetrandrine is the following: 1) tetrandrine inhibits the MDR pump by stimulating MDR ATPase which limits the energy of the pump by depletion of parasite ATP, 2) tetrandrine blocks the genetic factor which controls the induction of the pump. Therefore, it appears that the parasite cannot outsmart these mechanisms and produce a new mode of resistance. Only time will tell if this is correct.

Glutathione Transport: A New Role for PfCRT in Chloroquine Resistance

Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV.

Optimization of 4-Aminoquinoline/Clotrimazole-Based Hybrid Antimalarials: Further Structure–Activity Relationships, in Vivo Studies, and Preliminary Toxicity Profiling

Despite recent progress in the fight against malaria, the emergence and spread of drug-resistant parasites remains a serious obstacle to the treatment of infections. We recently reported the development of a novel antimalarial drug that combines the 4-aminoquinoline pharmacophore of chloroquine with that of clotrimazole-based antimalarials. Here we describe the optimization of this class of hybrid drug through in-depth structure-activity relationship studies. Antiplasmodial properties and mode of action were characterized in vitro and in vivo, and interactions with the parasite's 'chloroquine resistance transporter' were investigated in a Xenopus laevis oocyte expression system. These tests indicated that piperazine derivatives 4b and 4d may be suitable for coadministration with chloroquine against chloroquine-resistant parasites. The potential for metabolism of the drugs by cytochrome P450 was determined in silico, and the lead compounds were tested for toxicity and mutagenicity. A preliminary pharmacokinetic analysis undertaken in mice indicated that compound 4b has an optimal half-life.