Identification Of Antimalarial Compounds And Their Mode Of Actions

Malaria is one of the most significant infectious diseases in the tropics, claiming around half-million lives annually, mainly due to Plasmodium falciparum (P. falciparum) infections. To date, P. falciparum has developed full or partial resistance towards all three classes of antimalarials, including against the artemisinin. Hence, it may jeopardize the efficacy of the current antimalarial treatment regimen, the Artemisinin Combination Therapy (ACT), in the near future. Therefore, there is an utmost need to identify a new antimalarial compound with a novel mode of action (MoA). This study mainly focused on identifying antimalarial compounds from soil microorganisms isolated from Malaysia and Japan. The crude extracts of these strains showed potent antimalarial activity in a preliminary assay against Pf 3D7 (wild type). The crude extracts of Malaysian strains also inhibited the activity of human GSK-3β (75 % similar to P. Falciparum GSK-3 (Pf GSK-3))

ATP gatekeeper of Plasmodium protein kinase may provide the opportunity to develop selective antimalarial drugs with multiple targets

Malaria is one of the most devastating infectious diseases that caused millions of clinical cases annually despite decades of prevention efforts. Recent cases of Plasmodium falciparum resistance against the only remaining class of effective antimalarial (artemisinin) in South East Asia may soon pose a significant threat. Hence, the identification of new antimalarial compounds with a novel mode of action is necessary to curb this problem. Protein kinase has been implicated as a valid target for drug development in diseases such as cancer and diabetes in humans. A similar approach is now recognized for the treatment of protozoan-related disease including malaria. Few Plasmodium protein kinases that are not only crucial for their survival but also have unique structural features have been identified as a potential target for drug development. In this review, studies on antimalarial drug development exploiting the size of Plasmodium protein kinase ATP gatekeeper over the past 15 years are mainly discussed. The ATP-binding site of Plasmodium protein kinases such as Pf CDPK1, Pf CDPK4, Pf PKG, Pf PK7, and Pf PI4K showed great potential for selective and multi-target inhibitions owing to their smaller or unique ATP-gatekeeper amino acid subunits compared to that of human protein kinase. Hence it is a feasible solution to identify a new class of active antimalarial agents with a novel mode of action and longer clinical life-span

Assessment of the inhibitory mechanism of action for a yeast cell-based screening system targeting glycogen synthase kinase-3ß (GSK-3ß)

Background and Objective: Glycogen Synthase Kinase-3 (GSK-3) is one of the prior targets for drug discovery due to its involvement in many cell signaling and metabolism. It has been implicated in several critical diseases such as diabetes, Alzheimer’s disease, cancer and inflammation. To date, many GSK-3 inhibitors have been identified and classified into different type such as inorganic atom, ATP competitive and non-ATP competitive types. Many laboratories worldwide are still actively screening for bioactive compounds for GSK-3 inhibitory activity using diverse screening systems. This study assessed an assay developed using a yeast cell-based system specifically targeting GSK-3β for preliminary screening and cost effectiveness. Methodology: In this study, the GSK-3 homologues in yeast (MCK1, MDS1, MRK1 and YOL128C) were knocked out and inserted with mammalian GSK-3β. In order to determine the inhibitory mechanism, known GSK-3β inhibitors were tested and evaluated. Results: The GSK-3β inhibitor I and staurosporine showed inhibition on GSK-3β activity at a concentration of 1 and 20 μg disc–1, respectively. Other known inhibitors, such as indirubin-3’-monoxime, kenpaullone, GSK-3 inhibitor IV and enzastaurin showed no detectable inhibition in this study. Conclusion: The GSK-3β inhibitor I and staurosporine interacted with the same amino acid on GSK-3β which is Cys199 while other inhibitors have no interactions with Cys199 as reported in docking study. This study suggests that this yeast cell-based system can be used to screen GSK-3β inhibitors that is targeting on Cys199 residue

Plasmodium falciparum protein kinase as a potential therapeutic target for antimalarial drugs development

Malaria is one of the most dangerous infectious diseases due to its high infection and mortality rates, especially in the tropical belt. Plasmodium falciparum (P. falciparum), the most virulent malaria parasite in humans, was recently reported to develop resistance against the final efficient antimalarial drug, artemisinin. Little is known about the resistance mechanisms, which further complicates the problem as a proper counteraction is unable to be taken. Hence, the understanding of drug mode of action and its molecular target is valuable knowledge that needs to be considered to develop the next generation of antimalarial drugs. P. falciparum protein kinase (Pf PK) is an attractive target for antimalarial chemotherapy due to its vital roles in all P. falciparum life stages. Moreover, overall structural differences and the presence of unique Pf PKs that are absent in human kinome, suggesting specific inhibition of Pf PK without affecting human cells is achievable. To date, at least 86 eukaryotic protein kinases have been identified in P. falciparum kinome, by which less than 40 were validated as potential targets at the erythrocytes stage. In this review, recent progress of the furthest validated Pf PKs; Pf Nek-1, Pf CDPK1, Pf CDPK4, Pf PKG, and Pf CLK-3 will be briefly discussed. © 2020, Malaysian Society for Parasitology. All rights reserved

Anti-malarial activities of two actinomycete isolates from sabah soil involved inhibition of glycogen synthase kinase 3ß

Exploiting natural resources for bioactive compounds is an attractive drug discovery strategy in search for new anti-malarial drugs with novel modes of action. Initial screening efforts in our laboratory revealed two preparations of soil-derived actinomycetes (H11809 and FH025) with potent anti-malarial activities. Both crude extracts showed glycogen synthase kinase 3β (GSK3β)-inhibitory activities in a yeast-based kinase assay. We have previously shown that the GSK3 inhibitor, lithium chloride (LiCl), was able to suppress parasitaemia development in a rodent model of malarial infection. The present study aims to evaluate whether anti-malarial activities of H11809 and FH025 involve the inhibition of GSK3β. The acetone crude extracts of H11809 and FH025 each exerted strong inhibition on the growth of Plasmodium falciparum 3D7 in vitro with 50% inhibitory concentration (IC50) values of 0.57 ± 0.09 and 1.28 ± 0.11 µg/mL, respectively. The tested extracts exhibited Selectivity Index (SI) values exceeding 10 for the 3D7 strain. Both H11809 and FH025 showed dosage-dependent chemo-suppressive activities in vivo and improved animal survivability compared to non-treated infected mice. Western analysis revealed increased phosphorylation of serine (Ser 9) GSK3β (by 6.79 to 6.83-fold) in liver samples from infected mice treated with H11809 or FH025 compared to samples from non-infected or non-treated infected mice. A compound already identified in H11809 (data not shown), dibutyl phthalate (DBP) showed active anti-plasmodial activity against 3D7 (IC50 4.87 ± 1.26 µg/mL which is equivalent to 17.50 µM) and good chemo-suppressive activity in vivo (60.80% chemo-suppression at 300 mg/kg body weight [bw] dosage). DBP administration also resulted in increased phosphorylation of Ser 9 GSK3β compared to controls. Findings from the present study demonstrate that the potent anti-malarial activities of H11809 and FH025 were mediated via inhibition of host GSK3β. In addition, our study suggests that DBP is in part the bioactive component contributing to the antimalarial activity displayed by H11809 acting through the inhibition of GSK3β

Bioactivities and mode of actions of dibutyl phthalates and nocardamine from Streptomyces sp. H11809

Dibutyl phthalate (DBP) produced by Streptomyces sp. H11809 exerted inhibitory activity against human GSK-3β (Hs GSK-3β) and Plasmodium falciparum 3D7 (Pf 3D7) malaria parasites. The current study aimed to determine DBP’s plausible mode of action against Hs GSK-3β and Pf 3D7. Molecular docking analysis indicated that DBP has a higher binding affinity to the substrate-binding site (pocket 2; −6.9 kcal/mol) than the ATP-binding site (pocket 1; −6.1 kcal/mol) of Hs GSK-3β. It was suggested that the esters of DBP play a pivotal role in the inhibition of Hs GSK-3β through the formation of hydrogen bonds with Arg96/Glu97 amino acid residues in pocket 2. Subsequently, an in vitro Hs GSK-3β enzymatic assay revealed that DBP inhibits the activity of Hs GSK-3β via mixed inhibition inhibitory mechanisms, with a moderate IC50 of 2.0 µM. Furthermore, the decrease in Km value with an increasing DBP concentration suggested that DBP favors binding on free Hs GSK-3β over its substrate-bound state. However, the antimalarial mode of action of DBP remains unknown since the generation of a Pf 3D7 DBP-resistant clone was not successful. Thus, the molecular target of DBP might be indispensable for Pf survival. We also identified nocardamine as another active compound from Streptomyces sp. H11809 chloroform extract. It showed potent antimalarial activity with an IC50 of 1.5 µM, which is ~10-fold more potent than DBP, but with no effect on Hs GSK-3β. The addition of ≥12.5 µM ferric ions into the Pf culture reduced nocardamine antimalarial activity by 90% under in vitro settings. Hence, the iron-chelating ability of nocardamine was shown to starve the parasites from their iron source, eventually inhibiting their growth

Draft genome sequence data of Streptomyces sp. FH025

The genome data of Streptomyces sp. FH025 comprised of 8,381,474 bp with a high GC content of 72.51%. The genome contains 7035 coding sequences spanning 1261 contigs. Streptomyces sp. FH025 contains 57 secondary metabolite gene clusters including polyketide synthase, nonribosomal polyketide synthase and other biosynthetic pathways such as amglyccycl, butyrolactone, terpenes, siderophores, lanthipeptide-class-iv, and ladderane. 16S rRNA analysis of Streptomyces sp. FH025 is similar to the Streptomyces genus. This whole genome project has been deposited at NCBI under the accession JAFLNG000000000

The isolation rate of culturable actinomycetes from Malaysian Borneo forests and their activity against mammalian GSK-3β

More than ten types of forests can be found in Sabah, Malaysian Borneo. Studies comparing culturable actinomycetes potential in this region are relatively scarce. This study described a preliminary statistical comparison of culturable actinomycetes isolation rates and their biological activity against mammalian glycogen synthase kinase-3 (GSK-3β). We isolated 1049 isolates using standard isolation media for actinomycetes (HVA, ISP4 and AIA) with distinctive morphologies from the main forest types in Sabah; primary, secondary, mangrove, and beach forests. Isolate prevalence analysis revealed that secondary forests had the highest soil-to-isolate ratio (1:11). Interestingly, Kruskal-Wallis analysis revealed no significant differences in the overall isolation rates of actinomycetes, including non-sporulating strains, between forest types (P-value=0.142). The crude extracts of these isolates were assayed against GSK-3β, and we identified 19 active isolates; nine from primary and nine from secondary forests (no significant mean difference (P-value=0.558), one from beach forests, and none from mangrove forests. Overall, despite the different sampling locations and soil types, the isolation rates of culturable actinomycetes in Sabah did not vary significantly. However, both primary and secondary forests yielded more actinomycetes isolates that were active against mammalian GSK-3β than mangrove and beach forests. Hence, secondary forests are an attractive alternative to primary forests for exploring bioactive compounds from culturable actinomycetes in Sabah