Anti-Malarial Activity of Goniothalamus Scortechinii King

Malaria remains the most devastating infectious parasitic disease, inflicting both death and economic loses on at least half the world population. Numerous attempts have been made to control the disease by using vector control measures or/and chemoprophylaxis, but they have had limited success. Immunoprophylaxis hold promises but effective vaccines are still not available. Presently, the most effective way of dealing with malaria is the administration of chemotherapeutic agents. Although drugs treatments of malaria are currently the best means of disease management, there is an urgent need for the development of effective anti-malarial drugs. Earlier assessment of Goniothalamus scortechinii King showed to possess significant anti-malarial properties, in vitro. A phytochemical study of G. schortechinii King was thus carried out and has led to the isolation and characterization of two compounds, goniothalamin and pinocembrine, from the bioactive chloroform fraction. Both compounds were assayed for anti-malarial activity using the pLDH method. Both exhibited anti-malarial activity against P. falciparum in different degrees, goniothalamin gave an IC50 value of 4.0824 μg/ml while pinocembrine gave 19.308 μg/ml. Goniothalamin was evaluated for its anti-malaria activity in-vivo using 4-Day Suppressive Test against Plasmodium berghei ANKA strain in Swiss Albino Mice. The 4DT was carried out by inoculating the clean mice with P berghei ANKA strain and the infected mice were then treated orally and subcutaneously with goniothalamin. The suppression of parasite parasitemia and the ED90 value of goniothalamin were determined. Control drug used in this study was Chloroquine. Results showed that goniothalamin when given orally at a dose of 90 and 120 mg/kg mice body weight, exhibited suppressions of P. berghei infection of 98% and 99.7%, respectively. Meanwhile, goniothalamin given subcutaneously at a dose 120 mg/kg mice body weight gave 90.5% suppression of P. berghei infection. Ex vivo assay was carried out to investigate the effect of goniothalamin towards P. falciparum in vitro using the mouse serum treated with goniothalamin. This was done to prove that goniothalamin reaction toward P. falciparum should same as reaction towards P. berghei in in vivo reaction. Ex vivo test was carried out using pLDH assay with serum of mice given goniothalamin orally and subcutaneously. A graph to determine the 90% inhibition of drugs-serum towards P. falciparum was plotted for each treated mice serum. Results showed the IS90 of mice serum given goniothalamin orally was ranging from 0.050 to 4.00 μg/ml, for subcutaneous route the IS90 was ranging from 0.009-4.750 μg/ml. A graph for estimating the length of time goniothalamin can remain in the blood was plotted. This gave the estimated time of goniothalamin both given orally and subcutaneously can remained a minimum of 6 hours in the blood. In conclusion, goniothalamin does strongly inhibit P. falciparum, although it is not as potent as the standard drugs in use. More investigations such as drug combination, cytotoxicity, mechanism of action and toxicology studies, need to be carried out in order to determine its full potential as an anti-malarial

Plasmodial enzymes in metabolic pathways as therapeutic targets and contemporary strategies to discover new antimalarial drugs: a review

Malaria continues to pose imminent threat to the world population, as the mortality rate associated with this disease remains high. Current treatment relies on antimalarial drugs such as Artemisinin Combination Therapy (ACT) are still effective throughout the world except in some places, where ACT-resistance has been reported, thus necessitating novel approaches to develop new anti-malarial therapy. In the light of emerging translational research, several plasmodial targets, mostly proteins or enzymes located in the parasite’s unique organelles, have been extensively explored as potential candidates for the development of novel antimalarial drugs. By targeting the metabolic pathways in mitochondrion, apicoplast or cytoplasm of Plasmodium, the possibility to discover new drugs is tremendous, as they have potentials as antimalarial therapeutic targets. This literature review summarizes pertinent information on plasmodial targets, especially enzymes involved in specific metabolic pathways, and the strategies used to discover new antimalarial drugs. © 2019, University of Malaya. All rights reserved

Evaluating the binding interactions between Artemisinin and Kelch 13 protein mutants via molecular modelling and docking studies

Malaria is a parasitic infection caused by protozoan parasites from the genus Plasmodium. Over the years, various concerns have arisen regarding the efficacy in treating malaria caused by Plasmodium falciparum, which was reported to be caused by mutations in one of the parasite’s proteins, known as the Kelch 13 (K13). This study aims to generate the model structures of P.falciparum K13 protein mutants and to evaluate the binding affinities and interactions between these proteins and artemisinin drug, which is the drug used for the treatment of malaria. To date, the interactions between the protein mutants and artemisinin drug have not been computationally elucidated. In this study, four different types of mutant proteins were analysed, which are V494I, L598G, S600C and N537I and the results were compared with the wild-type K13 protein. Homology models of these proteins were created using the wild-type K13 (PDB ID:4YY8), with high percentage of sequence identity with the mutants. Most models with -2 and 2 have good Rama-Z scores, hence it can be deduced that the four mutants V494I (-1.21 ± 0.42), L598G (-1.19 ± 0.41), S600C (- 0.93 ± 0.43), N537I (-1.16 ± 0.43) and the wild-type (-1.34 ± 0.45) have acceptable Rama-Z scores. Molecular docking between artemisinin and the generated models of K13 proteins revealed that all protein mutants have higher binding energy; V494I (-6.79 kcal/mol), L598G (-9.26 kcal/mol), S600C (-6.17 kcal/mol) and N537I (-6.96 kcal/mol), compared to the wildtype (-9.65 kcal/mol). The results showed that all four distinct mutant proteins have less stable complex formation, which indicate that the mutant proteins have higher resistance towards artemisinin due to the higher binding energy compared to the K13 wild-type protein. However, all mutations have a higher number of protein-ligand hydrophobic interactions and protein-ligand hydrogen bonds than the wild-type protein, which requires further analysis to understand the binding interactions. The predicted structural information with regards to binding interactions between the K13 mutant proteins and artemisinin obtained from this study has paved the path toward understanding how mutants may cause parasites’ resistance towards artemisinin drug

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum.

The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum. Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed. The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.This work was funded by projects PI17/01791 and PI17CIII/00035 from the Instituto de Salud Carlos III (Ministry of Science and Innovation) and cofounded by the European Regional Development Fund. AMR is financed with an ISCIII- RIO HORTEGA contract (AESI RRHH-2017) from the Instituto de Salud Carlos III (Ministry of Science and Innovation). MJB is financed with a project associated contract from the Instituto de Salud Carlos III (Ministry of Science and Innovation)S