Comparative study on mitogen activated protein kinase of Plasmodium species by using in silico method / Mohd Fakharul Zaman Raja Yahya and Hasidah Mohd Sidek

Malaria parasites, Plasmodium can infect a wide range of hosts including humans and rodents. There are two copies of mitogen activated protein kinases (MAPKs) in Plasmodium, namely MAPK1 and MAPK2. The MAPKs have been studied extensively in the human Plasmodium, P. falciparum. However, the MAPKs from other Plasmodium species have not been characterized and it is therefore the premise of presented study to characterize the MAPKs from other Plasmodium species-P. vivax, P. knowlesi, P. berghei, P. chabaudi and P.yoelli using a series of publicly available bioinformatic tools. In silico data indicates that all Plasmodium MAPKs are nuclear-localizedandcontain both a nuclear localization signal (NLS) anda Leucine-rich nuclear export signal (NES). The activation motifs ofTDYand TSH werefound to befully conserved in Plasmodium MAPK1 and MAPK2, respectively

Pencirian molekul glikogen sintase kinase-3 dari Eimeria tenella

Penemuan sasaran dadah antikoksidia baharu merupakan antara usaha yang diperlukan untuk mengawal penyakit koksidiosis ayam yang disebabkan oleh spesies Eimeria. Dalam kajian ini, serpihan yang mengekodkan glikogen sintase kinase-3 (GSK-3) Eimeria tenella putatif telah diamplifikasi daripada cDNA E. tenella. Hasil pemadanan homologi menunjukkan jujukan GSK-3 E. tenella yang terjana mempunyai padanan yang tinggi dengan jujukan GSK-3 organisma lain. Domain terpulihara GSK-3 dan residu yang penting untuk aktiviti GSK-3 juga diramalkan hadir dalam jujukan GSK-3 E. tenella. Analisis struktur sekunder serta pemodelan homologi menunjukkan pembahagian struktur protein kepada domain bebenang beta pada hujung N dan domain heliks alfa pada hujung C, yang merupakan ciri enzim GSK-3. Kesemua hasil analisis ini menyokong bahawa jujukan yang dikaji mengekodkan protein GSK-3 dalam E. tenella. Walaupun darjah keterpuliharaan adalah tinggi, namun terdapat perbezaan yang bermakna diperhatikan antara GSK-3 E. tenella dan perumahnya. Residu Ser 9 yang dilaporkan penting untuk perencatan aktiviti GSK-3 didapati tidak terpulihara dalam GSK-3 E. tenella. Memandangkan Ser 9 merupakan tapak pemfosfatan bagi GSK-3β dalam haiwan vertebrata, ketiadaan residu ini dalam jujukan GSK-3 E. tenella mencadangkan bahawa pengawalaturan GSK-3 E. tenella melibatkan tapak pemfosfatan dan mekanisme yang berbeza. Tambahan pula, hasil analisis filogenetik menunjukkan bahawa GSK-3 E. tenella mempunyai pertalian yang rapat dengan protein GSK-3 tumbuh-tumbuhan. Analisis superposisi GSK-3 E. tenella dengan GSK-3β Homo sapiens pula menunjukkan bahawa perencat GSK-3 mampu berinteraksi dengan protein GSK-3 E. tenella. Keputusan kajian ini mencadangkan bahawa GSK-3 E. tenella mempunyai potensi untuk diperkembangkan sebagai sasaran dadah antikoksidia

The antimalarial effect of curcumin is mediated by the inhibition of glycogen synthase kinase-3β

Curcumin, a bioactive compound in Curcuma longa, exhibits various pharmacological activities, including antimalarial effects. In silico docking simulation studies suggest that curcumin possesses glycogen synthase kinase-3β (GSK3β)-inhibitory properties. The involvement of GSK3 in the antimalarial effects in vivo is yet to be demonstrated. In this study, we aimed to evaluate whether the antimalarial effects of curcumin involve phosphorylation of host GSK3β. Intraperitoneal administration of curcumin into Plasmodium berghei NK65-infected mice resulted in dose-dependent chemosuppression of parasitemia development. At the highest dose tested (30 mg/kg body weight), both therapeutic and prophylactic administrations of curcumin resulted in suppression exceeding 50% and improved median survival time of infected mice compared to control. Western analysis revealed a 5.5-fold (therapeutic group) and 1.8-fold (prophylactic group) increase in phosphorylation of Ser 9 GSK3β and 1.6-fold (therapeutic group) and 1.7-fold (prophylactic group) increase in Ser 473 Akt in liver of curcumin-treated infected animals. Following P. berghei infection, levels of pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-10, and IL-4 were elevated by 7.5-, 35.0-, 33.0-, and 2.2-fold, respectively. Curcumin treatment (therapeutic) caused a significant decrease (by 6.0- and 2.0-fold, respectively) in serum TNF-α and IFN-γ level, while IL-10 and IL-4 were elevated (by 1.4- and 1.8-fold). Findings from the present study demonstrate for the first time that the antimalarial action of curcumin involved inhibition of GSK3β

Antiplasmodium and chloroquine resistance reversing effects of embelin against Plasmodium falciparum K1

Background: Emergence of chloroquine (CQ) resistance among different strains of Plasmodium falciparum is the worst catastrophe that has ever perplexed efforts to eradicate malaria. This behooved the scientists to search for new alternatives or sensitizers that augment its action. Method: In this experiment, the potential of embeline, a quinonoid phytochemical obtained from the Indian plant Embelia ribes, to inhibit the growth and sensitize CQ action was screened using SYBRE green-1 based drug sensitivity assay and isobologram technique respectively. Its effect on both RBCs and Vero cells stability and RBCs fragility was screened to assess its safety. To depict its molecular mechanism, its effect on hemozoin formation and the new permeation pathway (NPP) of the host RBCs membrane were screened. Furthermore, its anti-oxidant activity was measured using the conventional in vitro tests and its molecular characters were obtained using Molispiration program. Results: The results showed that its antiplasmodial effect was weaker than CQ but synergism was obtained when they were combined together. Its low anti-plasmodial potency and poor selectivity toward RBCs reduces its eligibility to be introduced as an antimalarial. Nevertheless, it synergized CQ at a concentration within the safe limit. Embelin did not produce any impact on NPP but its impact on hemozoin formation was pronounced. Its antiplasmodium effect can be attributed to the latter or to its effect on RBCs membrane but further investigations are required to detect its CQ resistance. Conclusion: Overall, embelin is not ideal to be used as antiplasmodium but can be suggested as CQ resistance reversing agent

Anti-malarial and anti-inflammatory effects of Gynura procumbens are mediated by kaempferol via inhibition of glycogen synthase kinase-3ß (GSK3ß)

Gynura procumbens is a medicinal plant, traditionally used to treat inflammation and fever. A yeast-based assay detected GSK3â-inhibitory activity in the aqueous extract of G. procumbens. GSK3â is now known to have a central role in the modulation of host inflammatory response during bacterial infections. In this study, we investigated the involvement of GSK3â in the anti-malarial and anti-inflammatory effects of an aqueous extract of G. procumbens. Our results showed that G. procumbens inhibited growth of P. falciparum 3D7. Consecutive four-day administration of 250 mg/kg body weight (b.w.) G. procumbens resulted in strong chemosuppression and improved survivability in P. berghei-infected mice. B. pseudomallei-infected mice treated with G. procumbens (50 mg/kg b.w.) showed increased survivability. TNF-á and IFN-ã levels in liver and serum of B. pseudomallei-infected mice were lowered by G. procumbens treatment. IL-10 level was higher in serum of G. procumbens-administered infected mice. G. procumbens treatment of P. berghei-and B. pseudomallei-infected animals each resulted in increased hepatic GSK3â (Ser9) phosphorylation. It is noteworthy that kaempferol (one of the compounds in G. procumbens) also inhibited the growth of P. falciparum 3D7; showed strong chemosuppression and improved survivability in P. berghei-infected mice at 5 mg/kg b.w. B. pseudomallei-infected mice treated with kaempferol (10 mg/kg b.w.) showed improved survivability. Concomitantly, the described effects due to kaempferol also involved enhanced GSK3â (Ser9) phosphorylation as observed with G. procumbens. In summary, the observed anti-malarial and anti-inflammatory effects of G. procumbens involved inhibition of GSK3â and kaempferol may in part be responsible for the pharmacological effects

Anti-malarial and anti-inflammatory effects of Gleichenia truncata mediated through inhibition of GSK3ß

Gleichenia truncata is a highland fern from the Gleicheniaceae family known for its traditional use among indigenous communities in Asia to treat fever. The scientific basis of its effect has yet to be documented. A yeast-based kinase assay conducted in our laboratory revealed that crude methanolic extract (CME) of G. truncata exhibited glycogen synthase kinase-3 (GSK3)-inhibitory activity. GSK3β is now recognized to have a pivotal role in the regulation of inflammatory response during bacterial infections. We have also previously shown that lithium chloride (LiCl), a GSK3 inhibitor suppressed development of Plasmodium berghei in a murine model of malarial infection. The present study is aimed at evaluating G. truncata for its anti-malarial and anti-inflammatory effects using in vivo malarial and melioidosis infection models respectively. In a four-day suppressive test, intraperitoneal injections of up to 250 mg/kg body weight (bw) G. truncata CME into P.berghei-infected mice suppressed parasitaemia development by >60%. Intraperitoneal administration of 150 mg/kg bw G. truncata CME into Burkholderia pseudomallei-infected mice improved survivability by 44%. G. truncata CME lowered levels of pro-inflammatory cytokines (TNF-α, IFN-γ) in serum and organs of B. pseudomallei-infected mice. In both infections, increased phosphorylations (Ser9) of GSK3β were detected in organ samples of animals administered with G. truncata CME compared to controls. Taken together, results from this study strongly suggest that the anti-malarial and anti-inflammatory effects elicited by G. truncata in part were mediated through inhibition of GSK3β. The findings provide scientific basis for the ethnomedicinal use of this fern to treat inflammation-associated symptoms

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

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

Andrographolide effect on both Plasmodium falciparum infected and non infected RBCs membranes

Objective: To explore whether its antiplasmodium effect of andrographolide is attributed to its plausible effect on the plasma membrane of both Plasmodium falciparum infected and non-infected RBCs. Methods: Anti-plasmodium effect of andrographolide against Plasmodium falciparum strains was screened using the conventional malaria drug sensitivity assay. The drug was incubated with uninfected RBCs to monitor its effect on their morphology, integrity and osmotic fragility. It was incubated with the plasmodium infected RBCs to monitor its effect on the parasite induced permeation pathways. Its effect on the potential of merozoites to invade new RBCs was tested using merozoite invasion assay. Results: It showed that at andrographolide was innocuous to RBCs at concentrations approach its therapeutic level against plasmodia. Nevertheless, this inertness was dwindled at higher concentrations. Conclusions: In spite of its success to inhibit plasmodium induced permeation pathway and the potential of merozoites to invade new RBCs, its anti-plasmodium effect can't be attributed to these functions as they were attained at concentrations higher than what is required to eradicate the parasite. Consequently, other mechanisms may be associated with its claimed actions

Pencirian jangkitan Plasmodium berghei NK65 pada mencit ICR sebagai model jangkitan malaria teruk

Malaria teruk atau ‘severe’ kebiasaannya disebabkan oleh jangkitan Plasmodium falciparum. Jangkitan Plasmodium falciparum pada manusia boleh menyebabkan kerosakan organ, anemia teruk, komplikasi serius, koma dan kematian. Bagi tujuan memahami patogenesis malaria teruk, model haiwan digunakan dalam kajian kali ini bagi mengenal pasti sama ada gabungan hos-parasit daripada mencit ICR dengan Plasmodium berghei NK65 boleh menyebabkan jangkitan malaria teruk pada hos. Pencirian jangkitan P. berghei ANKA pernah dilakukan sebelum ini terhadap mencit ICR; walau bagaimanapun, pencirian jangkitan P. berghei NK65 secara terperinci terhadap mencit ICR dalam kajian ini adalah pertama kali dilaporkan. Inokulasi sel darah merah (RBC) terjangkit-P. berghei NK65 (2 × 107 parasit RBC (pRBC)/ mL) dilakukan terhadap mencit ICR dengan suntikan secara intraperitoneum. Pemantauan perubahan ciri fizikal seperti berat, suhu mencit, kematian mencit, pos mortem, histologi dan aras sitokin inflamasi yang terhasil selepas jangkitan direkod untuk analisis. Strain P. berghei NK65 menghasilkan jangkitan tahap teruk terhadap mencit ICR iaitu paras parasitemia melebihi 50% pada hari ke-10 selepas jangkitan diikuti kematian. Analisis histopatologi menunjukkan jangkitan ini menyebabkan perubahan pada tisu serebrum, perlekatan leukosit pada endotelium dan pensekuesteran pRBC dalam salur darah serebrum serta pendarahan intravaskular. Selepas jangkitan, pensekuesteran pRBC dan pengumpulan pigmen malaria turut dilihat pada organ utama mencit. Tambahan lagi, edema pulmonari, pembentukan membran hialin pada peparu dan pendarahan kortikal pada ginjal dilihat pada mencit terjangkit. Sitokin proinflamasi (TNF-α, IFN-γ, dan IL-18) dan sitokin antiinflamasi (IL-10 dan IL-4) juga meningkat dalam serum mencit terjangkit. Secara rumusannya, model jangkitan mencit ICR-P. berghei NK65 yang digunakan dalam kajian ini menunjukkan ciri-ciri jangkitan malaria teruk. Hasil daripada kajian ini boleh digunakan sebagai asas untuk memahami patogenesis bagi malaria teruk pada manusia dan model jangkitan malaria haiwan pada masa akan datang