Expression of the Fab enzymes (Fab I and Fab Z) from Plasmodium falciparum after exposure to Artemisia afra plant extracts and drugs screening.

The emergence and spread of drug resistance of the malaria parasite to the main treatment emphasize the need to develop new antimalarial drugs. In this context, the fatty acid biosynthesis (FAS_II) pathway of the malaria parasite is one of the ideal targets due to its crucial role in parasite survival. In this study, we report the expression and the affinity binding of Fab_I and Fab_Z after exposure to the parasite with different extracts of the Artemisia afra. The parasites were exposed for 2 days to different extracts. Gene expression was done to determine the level of expression of the fab enzymes after treatments. A GCMS was run to determine the different compounds of the plant extracts, followed by a virtual screening between the fab enzymes and the active compounds using Pyrex. The results showed different expression patterns of the Fab enzymes. Fab_I expression was downregulated in the W2 and D6 strains by the ethanolic extract but was increased by Hexane and DCM extracts. A different expression pattern was observed for Fab_Z. It was all upregulated except in the D6 strain when exposed to the ethanolic and hexane extracts. Virtual screening showed an affinity with many compounds. Hits compounds with high binding energy were detected. 11alphaHydroxyprogesterone and Aspidospermidin-17-ol were found to have high binding energy with Fab_I respectively (- 10.7 kcal/mol; - 10.2 kcal/mol). Fab_Z shows also high affinity with 11alpha-Hydroxyprogesterone (- 10 kcal/mol) and Thiourea (- 8.4 kcal/mol). This study shows the potential of A. afra to be used as a new source of novel antimalarial compounds.

Efficacy of artemisinin-lumefantrine for treatment of uncomplicated malaria after more than a decade of its use in Kenya.

BACKGROUND: The resistance of Plasmodium falciparum to antimalarial drugs remains a major impairment in the treatment and eradication of malaria globally. Following the introduction of artemisinin-based combination therapy (ACT), there have been reports of delayed parasite clearance. In Kenya, artemether-lumefantrine (AL) is the recommended first-line treatment of uncomplicated malaria. This study sought to assess the efficacy of AL after a decade of use as the preferred method of managing malarial infections in Kenya. We assessed clinical and parasitological responses of children under 5 years between May and November 2015 in Chulaimbo sub-County, Kisumu, Kenya. Patients aged between 6 and 60 months with uncomplicated P. falciparum mono-infection, confirmed through microscopy, were enrolled in the study. The patients were admitted at the facility for 3 days, treated with a standard dose of AL, and then put under observation for the next 28 days for the assessment of clinical and parasitological responses. Of the 90 patients enrolled, 14 were lost to follow-up while 76 were followed through to the end of the study period. Seventy-five patients (98.7%) cleared the parasitaemia within a period of 48 h while one patient (1.3%) cleared on day 3. There was 100% adequate clinical and parasitological response. All the patients cleared the parasites on day 3 and there were no re-infections observed during the stated follow-up period. This study, therefore, concludes that AL is highly efficacious in clearing P. falciparum parasites in children aged ≥6 and ≤60 months. The study, however, underscores the need for continued monitoring of the drug to forestall both gradual ineffectiveness and possible resistance to the drug in all target users.

Chemical Synthesis, Efficacy, and Safety of Antimalarial Hybrid Drug Comprising of Sarcosine and Aniline Pharmacophores as Scaffolds.

Malaria is a disease caused by protozoans transmitted to humans by infected female Anopheles mosquitoes. According to the WHO report of 2015, there were 214 million cases of malaria with 438,000 deaths worldwide. Ninety percent of world's malaria cases occur in Africa, where the disease is recognized as a serious impediment to economic and social development. Despite advancement in malaria research, the disease continues to be a global problem, especially in developing countries. Currently, there is no effective vaccine for malaria control. In addition, although there are effective drugs for treatment of malaria, this could be lost to the drug resistance in different Plasmodium species. The most lethal form is caused by P. falciparum which has developed resistance to many chemotherapeutic agents and possibly to the current drugs of choice. Reducing the impact of malaria is a key to achieving the sustainable development goals which are geared toward combating the disease. Covalent bitherapy is a rational and logical way of drug design which entails joining a couple of molecules with individual intrinsic action into a unique agent, hence packaging dual activity into one hybrid. This suggests the need to develop new antimalarial drugs that are effective against malaria parasites based on the new mode of action, molecular targets, and chemical structures. In silico studies have shown that sarcosine is able to bind to unique plasmodia proteins (P. falciparum ATCase), whereas aniline can be a ligand to target protein (enoyl acyl carrier protein reductase), hence suppressing the progression of the disease. The main objective of this study was to synthesize and determine the efficacy and safety of antiplasmodial hybrid drug comprising the sarcosine and aniline derivative for management of plasmodial infections. The hybrid drug was synthesized by adding thionyl chloride to sarcosine to form acyl chloride which was then added to aniline to form sarcosine-aniline hybrid molecule. The IC50 of sarcosine-aniline hybrid was 44.80 ± 4.70 ng/ml compared with that of aniline derivative which was 22.86 ± 1.26 ng/ml. The IC50 of control drugs was 2.63 ± 0.38 ng/ml and 5.69 ± 0.39 ng/ml for artesunate and chloroquine, respectively. There was a significant difference between IC50 of sarcosine-aniline hybrid and aniline derivative (p < 0.05). There was also a significant difference between sarcosine-aniline hybrid and standard drugs used to treat malaria including artesunate and chloroquine (p < 0.05). The ED50 of sarcosine-aniline hybrid drug was 6.49 mg/kg compared with that of aniline derivative which was 3.61 mg/kg. The ED50 of control drugs was 3.56 mg/kg, 2.94 mg/kg, and 1.78 mg/kg for artesunate-aniline hybrid, artesunate, and chloroquine, respectively. There was a significant difference (p < 0.05) between ED50 of sarcosine-aniline hybrid and both controls such as aniline derivative, artesunate, artesunate-aniline hybrid, and chloroquine. Cytotoxicity results revealed that sarcosine-aniline hybrid was safe to vero cells with a CC50 of 50.18 ± 3.53 µg/ml. Sarcosine-aniline hybrid was significantly less toxic compared with artesunate, chloroquine, and doxorubicin. Sarcosine-aniline hybrid was efficacious and safe to mice. Therefore, covalent bitherapy should be used in drug development for drug resistance mitigation.

Prevalence of mutations in Plasmodium falciparum genes associated with resistance to different antimalarial drugs in Nyando, Kisumu County in Kenya.

Resistance to the mainstay antimalarial drugs is a major concern in the control of malaria. Delayed Plasmodium falciparum parasite clearance has been associated with Single Nucleotide Polymorphisms (SNPs) in the kelch propeller region (K13). However, SNPs in the Pf-adaptor protein complex 2 mu subunit (Pfap2-mu), Pfcrt and Pfmdr1 are possible markers associated with multi-drug resistance. Here, we explored the prevalence of SNPs in the K13, Pfap2-mu, Pfcrt, and Pfmdr1 in 94 dried blood spot field isolates collected from children aged below 12 years infected with P. falciparum during a cross-sectional study. The samples were collected in 2015 during the peak malaria transmission season in the Nyando region of Western Kenya before treatment with Artemether-Lumefantrine, the first-line artemisinin-based combination therapy (ACT) in Kenya. However, 47 of the 94 samples had recurrent parasitemia and were interrogated for the presence of the SNPs in K13 and Pfap2-mu. We used PCR amplification and sequencing to evaluate specific regions of K13 (codons 432-702), Pfap2-mu (codons 1-350), Pfmdr1 (codons 86, 1034-1246), and Pfcrt (codons 72-76) gene(s). The majority of parasites harbored the wild type K13 sequence. However, we found a unique non-synonymous W611S change. In silico studies on the impact of the W611S predicted structural changes in the overall topology of the K13 protein. Of the 47 samples analyzed for SNPs in the Pfap2-mu gene, 14 (29%) had S160 N/T mutation. The CVIET haplotype associated with CQ resistance in the Pfcrt yielded a 7.44% (7/94), while CVMNK haplotype was at 92.56%. Mutations in the Pfmdr1 region were detected only in three samples (3/94; 3.19%) at codon D1246Y. Our data suggest that parasites in the western part of Kenya harbor the wildtype strains. However, the detection of the unique SNP in K13 and Pfap2-mu linked with ACT delayed parasite clearance may suggest slow filtering of ACT-resistant parasites.

Efficacy and safety evaluation of a novel trioxaquine in the management of cerebral malaria in a mouse model.

BACKGROUND: The emergence of multidrug-resistant strains of Plasmodium falciparum poses a great threat of increased fatalities in cases of cerebral and other forms of severe malaria infections in which parenteral artesunate monotherapy is the current drug of choice. The study aimed to investigate in a mouse model of human cerebral malaria whether a trioxaquine chemically synthesized by covalent linking of a 4,7-dichloroquinoline pharmacophore to artesunate through a recent drug development approach termed 'covalent bitherapy' could improve the curative outcomes in cerebral malaria infections. METHODS: Human cerebral malaria rodent model, the C57BL/6 male mice were infected intraperitoneally (ip) with Plasmodium berghei ANKA and intravenously (iv) treated with the trioxaquine from day 8 post-infection (pi) at 12.5 and 25 mg/kg, respectively, twice a day for 3 days. Treatments with the trioxaquine precursors (artesunate and 4,7-dichloroquine), and quinine were also included as controls. In vivo safety evaluation for the trioxaquine was done according to Organization for Economic Co-operation and Development (OECD) guidelines 423, where female Swiss albino mice were orally administered with either 300 or 2000 mg/kg of the trioxaquine and monitored for signs of severity, and or mortality for 14 days post-treatment. RESULTS: The trioxaquine showed a potent and a rapid antiplasmodial activity with 80% parasite clearance in the first 24 h for the two dosages used. Long-term parasitaemia monitoring showed a total parasite clearance as the treated mice survived beyond 60 days post-treatment, with no recrudescence observed. Artesunate treated mice showed recrudescence 8 days post-treatment, with all mice in this group succumbing to the infection. Also, 4,7-dichloroquinoline and quinine did not show any significant parasitaemia suppression in the first 24 h post-treatment, with the animals succumbing to the infection. CONCLUSION: Covalent bitherapy proves to be a viable source of urgently needed new anti-malarials for management of cerebral malaria, and this polypharmacology approach could be a potential strategy to protect artesunate from parasite resistance and in potentially improving clinical outcomes in severe forms of malaria infections.

In vitro selection of Plasmodium falciparum Pfcrt and Pfmdr1 variants by artemisinin.

BACKGROUND: Anti-malarial drugs are the major focus in the prevention and treatment of malaria. Artemisinin-based combination therapy (ACT) is the WHO recommended first-line treatment for Plasmodium falciparum malaria across the endemic world. Also ACT is increasingly relied upon in treating Plasmodium vivax malaria where chloroquine is failing. The emergence of artemisinin drug-resistant parasites is a serious threat faced by global malaria control programmes. Therefore, the success of treatment and intervention strategies is highly pegged on understanding the genetic basis of resistance. METHODS: Here, resistance in P. falciparum was generated in vitro for artemisinin to produce levels above clinically relevant concentrations in vivo, and the molecular haplotypes investigated. Genomic DNA was extracted using the QIAamp mini DNA kit. DNA sequences of Pfk13, Pfcrt and Pfmdr1 genes were amplified by PCR and the amplicons were successfully sequenced. Single nucleotide polymorphisms were traced by standard bidirectional sequencing and reading the transcripts against wild-type sequences in Codon code Aligner Version 5.1 and NCBI blast. RESULTS: Exposure of parasite strains D6 and W2 to artemisinin resulted in a decrease in parasite susceptibility to artemisinin (W2 and D6) and lumefantrine (D6 only). The parasites exhibited elevated IC50s to multiple artemisinins, with >twofold resistance to artemisinin; however, the resistance index obtained with standard methods was noticeably less than expected for parasite lines recovered from 50 µg/ml 48 h drug pressure. The change in parasite susceptibility was associated with Pfmdr-185K mutation, a mutation never reported before. The Pfcrt-CVMNK genotype (Pfcrt codons 72-76) was retained and notably, the study did not detect any polymorphisms reported to reduce P. falciparum susceptibility in vivo in the coding sequences of the Pfk13 gene. DISCUSSION: This data demonstrate that P. falciparum has the capacity to develop resistance to artemisinin derivatives in vitro and that this phenotype is achieved by mutations in Pfmdr1, the genetic changes that are also underpinning lumefantrine resistance. This finding is of practical importance, because artemisinin drugs in Kenya are used in combination with lumefantrine for the treatment of malaria. CONCLUSION: Artemisinin resistance phenotype as has been shown in this work, is a decrease in parasites susceptibility to artemisinin derivatives together with the parasite's ability to recover from drug-induced dormancy after exposure to drug dosage above the in vivo clinical concentrations. The study surmises that Pfmdr1 may play a role in the anti-malarial activity of artemisinin.

Chloroquine sensitivity: diminished prevalence of chloroquine-resistant gene marker pfcrt-76 13 years after cessation of chloroquine use in Msambweni, Kenya.

BACKGROUND: Plasmodium falciparum resistance to chloroquine (CQ) denied healthcare providers access to a cheap and effective anti-malarial drug. Resistance has been proven to be due to point mutations on the parasite's pfcrt gene, particularly on codon 76, resulting in an amino acid change from lysine to threonine. This study sought to determine the prevalence of the pfcrt K76T mutation 13 years after CQ cessation in Msambweni, Kenya. METHODS: Finger-prick whole blood was collected on 3MM Whatman(®) filter paper from 99 falciparum malaria patients. Parasite DNA was extracted via the Chelex method from individual blood spots and used as template in nested PCR amplification of pfcrt. Apo1 restriction enzyme was used to digest the amplified DNA to identify the samples as wild type or sensitive at codon 76. Prevalence figures of the mutant pfcrt 76T gene were calculated by dividing the number of samples bearing the mutant gene with the total number of samples multiplied by 100 %. Chi square tests were used to test the significance of the findings against previous prevalence figures. RESULTS: Out of 99 clinical samples collected in 2013, prevalence of the mutant pfcrt 76T gene stood at 41 %. CONCLUSION: The results indicate a significant [χ(2) test, P ≤ 0.05 (2006 vs 2013)] reversal to sensitivity by the P. falciparum population in the study site compared to the situation reported in 2006 at the same study site. This could primarily be driven by diminished use of CQ in the study area in line with the official policy. Studies to establish prevalence of the pfcrt 76T gene could be expanded countrywide to establish the CQ sensitivity status and predict a date when CQ may be re-introduced as part of malaria chemotherapy.

Fitness cost of resistance for lumefantrine and piperaquine-resistant Plasmodium berghei in a mouse model.

BACKGROUND: The evolution of drug-resistant parasites is a major hindrance to malaria control, and thus understanding the behaviour of drug-resistant mutants is of clinical relevance. The study aimed to investigate how resistance against lumefantrine (LU) and piperaquine (PQ), anti-malarials used as partner drugs in artemisinin-based combination therapy (ACT), impacts parasite fitness. This is important since resistance to ACT, the first-line anti-malarial regimen is increasingly being reported. METHODS: The stability of Plasmodium berghei ANKA strain that was previously selected for LU and PQ resistance was evaluated using the 4-day assay and established infection test in mice. Fitness cost of resistance was determined by comparing parasites proliferation rates in absence of drug pressure for the drug-exposed parasites between day 4 and 7 post-infection (pi), relative to the wild-type. Statistical analysis of data to compare mean parasitaemia and growth rates of respective parasite lines was carried out using student's t-test and one-way analysis of variance, with significance level set at p<0.05. RESULTS: During serial passaging in the absence of the drug, the PQ-resistant parasite maintained low growth rates at day 7 pi (mean parasitaemia, 5.6% ± 2.3) relative to the wild-type (28.4% ± 6.6), translating into a fitness cost of resistance of 80.3%. Whilst resistance phenotype for PQ was stable, that of LU was transient since after several serial passages in the absence of drug, the LU-exposed line assumed the growth patterns of the wild-type. CONCLUSIONS: The contrasting behaviour of PQ- and LU-resistance phenotypes support similar findings which indicate that even for drugs within the same chemical class, resistance-conferred traits may vary on how they influence parasite fitness and virulence. Resistance-mediating polymorphisms have been associated with less fit malaria parasites. In the absence of drug pressure in the field, it is therefore likely that the wild-type parasite will out-compete the mutant form. This implies the possibility of reintroducing a drug previously lost to resistance, after a period of suspended use. Considering the recent reports of high failure rates associated with ACT, high fitness cost of resistance to PQ is therefore of clinical relevance as the drug is a partner in ACT.

Novel drug targets in malaria parasite with potential to yield antimalarial drugs with long useful therapeutic lives.

The status of chemotherapy as the main strategy in malaria control is rapidly being eroded by development of drug resistant Plasmodia, causing malaria to be dubbed a "re-emerging disease". To counter this misfortune, there is an urgent need to develop novel antimalarial drugs capable of delaying resistance, or circumventing it altogether. Mode of action of antimalarial drugs, inter alia, has a bearing on their useful therapeutic lives (UTLs), with single target drugs having short UTLs compared with drugs which possess pleiotropic action. Quinolines and artemisinins are the two classes of drugs with pleiotropic action and subsequently long UTLs. All other antimalarials are single-target drugs, and have been rendered ineffective within 1 to 5 years of their introduction for clinical use. This strongly underlines the need for development of new antimalarial therapies possessing long UTLs. The present review explores novel drug targets within the malaria parasite that may be exploited in the search for novel drugs that possess long and UTLs.