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Öğe Alpha-Mangostin and its nano-conjugates induced programmed cell death in Acanthamoeba castellanii belonging to the T4 genotype(Springer, 2023) Ahmed, Usman; Ong, Seng-Kai; Tan, Kuan Onn; Khan, Khalid Mohammed; Khan, Naveed Ahmed; Siddiqui, Ruqaiyyah; Alawfi, Bader SaleemAcanthamoeba are free living amoebae that are the causative agent of keratitis and granulomatous amoebic encephalitis. Alpha-Mangostin (AMS) is a significant xanthone; that demonstrates a wide range of biological activities. Here, the anti-amoebic activity of alpha-Mangostin and its silver nano conjugates (AMS-AgNPs) were evaluated against pathogenic A. castellanii trophozoites and cysts in vitro. Amoebicidal assays showed that both AMS and AMS-AgNPs inhibited the viability of A. castellanii dose-dependently, with an IC50 of 88.5 +/- 2.04 and 20.2 +/- 2.17 mu M, respectively. Both formulations inhibited A. castellanii-mediated human keratinocyte cell cytopathogenicity. Functional assays showed that both samples caused apoptosis through the mitochondrial pathway and reduced mitochondrial membrane potential and ATP production, while increasing reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome-c reductase in the cytosol. Whole transcriptome sequencing of A. castellanii showed the expression of 826 genes, with 447 genes being up-regulated and 379 genes being down-regulated post treatment. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the majority of genes were linked to apoptosis, autophagy, RAP1, AGE-RAGE and oxytocin signalling pathways. Seven genes (PTEN, H3, ARIH1, SDR16C5, PFN, glnA GLUL, and SRX1) were identified as the most significant (Log2 (FC) value 4) for molecular mode of action in vitro. Future in vivo studies with AMS and nanoconjugates are needed to realize the clinical potential of this work.Öğe Anti-amoebic effects of synthetic acridine-9(10H)-one against brain-eating amoebae(Elsevier, 2023) Ahmed, Usman; Manzoor, Mehwish; Qureshi, Sehrish; Mazhar, Muzna; Fatima, Arj; Aurangzeb, Sana; Hamid, MehwishPathogenic A. castellanii and N. fowleri are opportunistic free-living amoebae. Acanthamoeba spp. are the causative agents of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK), whereas Naegleria fowleri causes a very rare but severe brain infection called primary amebic meningoencephalitis (PAM). Acridinone is an important heterocyclic scaffold and both synthetic and naturally occurring derivatives have shown various valuable biological properties. In the present study, ten synthetic Acridinone derivatives (I-X) were synthesized and assessed against both amoebae for anti-amoebic and cysticidal activities in vitro. In addition, excystation, encystation, cytotoxicity, host cell pathogenicity was also performed in-vitro. Furthermore, molecular docking studies of these compounds with three cathepsin B paralogous enzymes of N. fowleri were performed in order to predict the possible docking mode with pathogen. Compound VII showed potent anti-amoebic activity against A. castellanii with IC50 53.46 mu g/mL, while compound IX showed strong activity against N. fowleri in vitro with IC50 72.41 mu g/mL. Compounds II and VII showed a significant inhibition of phenotypic alteration of A. castellanii, while compound VIII significantly inhibited N. fowleri cysts. Cytotoxicity assessment showed that these compounds caused minimum damage to human keratinocyte cells (HaCaT cells) at 100 mu g/mL, while also effectively reduced the cytopathogenicity of Acanthamoeba to HaCaT cells. Moreover, Cathepsin B protease was investigated in-silico as a new molecular therapeutic target for these compounds. All compounds showed potential interactions with the catalytic residues. These results showed that acridine-9(10H)-one derivatives, in particular compounds II, VII, VIII and IX hold promise in the development of therapeutic agents against these free-living amoebae.Öğe Effect of embelin on inhibition of cell growth and induction of apoptosis in Acanthamoeba castellanii(Springer, 2023) Ahmed, Usman; Ong, Seng-Kai; Khan, Khalid Mohammed; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed; Shaikh, Mohd Farooq; Alawfi, Bader SaleemAcanthamoeba castellanii is the causative agent of fatal encephalitis and blinding keratitis. Current therapies remain a challenge, hence there is a need to search for new therapeutics. Here, we tested embelin (EMB) and silver nanoparticles doped with embelin (EMB-AgNPs) against A. castellanii. Using amoebicidal assays, the results revealed that both compounds inhibited the viability of Acanthamoeba, having an IC50 of 27.16 +/- 0.63 and 13.63 +/- 1.08 mu M, respectively, while causing minimal cytotoxicity against HaCaT cells in vitro. The findings suggest that both samples induced apoptosis through the mitochondria-mediated pathway. Differentially expressed genes analysis showed that 652 genes were uniquely expressed in treated versus untreated cells, out of which 191 were significantly regulated in the negative control vs. conjugate. Combining the analysis, seven genes (ARIH1, RAP1, H3, SDR16C5, GST, SRX1, and PFN) were highlighted as the most significant (Log2 (FC) value +/- 4) for the molecular mode of action in vitro. The KEGG analysis linked most of the genes to apoptosis, the oxidative stress signaling pathway, cytochrome P450, Rap1, and the oxytocin signaling pathways. In summary, this study provides a thorough framework for developing therapeutic agents against microbial infections using EMB and EMB-AgNPs.Öğe Lactase can target cellular differentiation of Acanthamoeba castellanii belonging to the T4 genotype(Springer, 2024) Simau, Fathimath Afaaf; Ahmed, Usman; Khan, Khalid Mohammed; Khan, Naveed Ahmed; Siddiqui, Ruqaiyyah; Alharbi, Ahmad M.; Alfahemi, HasanThe free living Acanthamoeba spp. are ubiquitous amoebae associated with potentially blinding disease known as Acanthamoeba keratitis (AK) and a fatal central nervous system infection granulomatous amoebic encephalitis (GAE). With the inherent ability of cellular differentiation, it can phenotypically transform to a dormant cyst form from an active trophozoite form. Acanthamoeba cysts are highly resistant to therapeutic agents as well as contact lens cleaning solutions. One way to tackle drug resistance against Acanthamoeba is by inhibiting the formation of cysts from trophozoites. The biochemical analysis showed that the major component of Acanthamoeba cyst wall is composed of carbohydrate moieties such as galactose and glucose. The disaccharide of galactose and glucose is lactose. In this study, we analyzed the potential of lactase enzyme to target carbohydrate moieties of cyst walls. Amoebicidal assessment showed that lactase was ineffective against trophozoite of A. castellanii but enhanced amoebicidal effects of chlorhexidine. The lactase enzyme did not show any toxicity against normal human keratinocyte cells (HaCaT) at the tested range. Hence, lactase can be used for further assessment for development of potential therapeutic agents in the management of Acanthamoeba infection as well as formulation of effective contact lens disinfectants.Öğe Malabaricones from the fruit of Myristica cinnamomea King as potential agents against Acanthamoeba castellanii(Elsevier, 2023) Ahmed, Usman; Sivasothy, Yasodha; Khan, Khalid Mohammed; Khan, Naveed Ahmed; Wahab, Siti Mariam Abdul; Awang, Khalijah; Othman, Muhamad AqmalAcanthamoeba castellanii is an opportunistic free-living amoeba (FLA) pathogen which can cause fatal central nervous system (CNS) infection, granulomatous amoebic encephalitis (GAE) and potentially blinding ocular infection, Acanthamoeba keratitis (AK). Acanthamoeba species remain a challenging protist to treat due to the unavailability of safe and effective therapeutic drugs and their ability to protect themselves in the cyst stage. Natural products and their secondary metabolites play a pivotal role in drug discovery against various pathogenic microorganisms. In the present study, the ethyl acetate extract of Myristica cinnamomea King fruit was evaluated against A. castellanii (ATCC 50492), showing an IC50 of 45.102 +/- 4.62 mu g/mL. Previously, the bio-guided fractionation of the extract resulted in the identification of three active compounds, namely Malabaricones (A-C). The isolated and thoroughly characterized acylphenols were evaluated for their anti-amoebic activity against A. castellanii for the first time. Among tested compounds, Malabaricone B (IC50 of 101.31 +/- 17.41 mu M) and Malabaricone C (IC50 of 49.95 +/- 6.33 mu M) showed potent anti-amoebic activity against A. castellanii trophozoites and reduced their viability up-to 75 and 80 %, respectively. Moreover, both extract and Malabaricones also significantly (p < 0.05) inhibit the encystation and excystation of A. castellanii, while showed minimal toxicity against human keratinocyte cells (HaCaT cells) at lower tested concentrations. Following that, the explanation of the possible mechanism of action of purified compounds were assessed by detection of the state of chromatin. Hoechst/PI 33342 double staining showed that necrotic cell death occurred in A. castellanii trophozoites after 8 h treatment of Malabaricones (A-C). These findings demonstrate that Malabaricones B and C could serve as promising therapeutic options against A. castellanii infections.Öğe Nanoparticle-Terpene Fusion: A Game-Changer in Combating Primary Amoebic Meningoencephalitis Caused by Naegleria fowleri(Amer Chemical Soc, 2024) Rajendran, Kavitha; Ahmed, Usman; Meunier, Alexia Chloe; Shaikh, Mohd Farooq; Siddiqui, Ruqaiyyah; Anwar, AyazPathogenic Naegleria fowleri (N. fowleri) are opportunistic free-living amoebae and are the causative agents of a very rare but severe brain infection called primary amoebic meningoencephalitis (PAM). The fatality rate of PAM in reported cases is more than 95%. Most of the drugs used againstN. fowleri infections are repurposed drugs. Therefore, a large number of compounds have been tested againstN. fowleri in vitro, but most of the tested compounds showed high toxicity and an inability to cross the blood-brain barrier. Andrographolide, forskolin, and borneol are important natural compounds that have shown various valuable biological properties. In the present study, the nanoconjugates (AND-AgNPs, BOR-AgNPs, and FOR-AgNPs) of these compounds were synthesized and assessed against both stages (trophozoite and cyst) ofN. fowleri for their antiamoebic and cysticidal potential in vitro. In addition, cytotoxicity and host cell pathogenicity were also evaluated in vitro. FOR-AgNPs were the most potent nanoconjugate and showed potent antiamoebic activity againstN. fowleriwith an IC50 of 26.35 mu M. Nanoconjugates FOR-AgNPs, BOR-AgNPs, and AND-AgNPs also significantly inhibit the viability of N. fowleri cysts. Cytotoxicity assessment showed that these nanoconjugates caused minimum damage to human keratinocyte cells (HaCaT cells) at 100 mu g/mL, while also effectively reducing the cytopathogenicity of N. fowleri trophozoites to the HaCaT cells. The outcomes of our experiments have unveiled substantial potential for AND-AgNPs, BOR-AgNPs, and FOR-AgNPs in the realm of developing innovative alternative therapeutic agents to combat infections caused by N. fowleri. This study represents a significant step forward in the pursuit of advanced strategies for managing such amoebic infections, laying the foundation for the development of novel and more effective therapeutic modalities in the fight against free-living amoebae.Öğe Natural Terpenes Inhibit the Cytopathogenicity of Naegleria fowleri Causing Primary Amoebic Meningoencephalitis in the Human Cell Line Model(Amer Chemical Soc, 2023) Rajendran, Kavitha; Ahmed, Usman; Meunier, Alexia Chloe; Shaikh, Mohd Farooq; Siddiqui, Ruqaiyyah; Anwar, AyazNaegleria fowleri is one of the free-living amoebae and is a causative agent of a lethal and rare central nervous system infection called primary amoebic meningoencephalitis. Despite the advancement in antimicrobial chemotherapy, the fatality rate in the reported cases is more than 95%. Most of the treatment drugs used against N. fowleri infection are repurposed drugs. Therefore, a large number of compounds have been tested against N. fowleri in vitro, but most of the compounds showed high toxicity. To overcome this, we evaluated the effectiveness of naturally occurring terpene compounds against N. fowleri. In this study, we evaluated the antiamoebic potential of natural compounds including Thymol, Borneol, Andrographolide, and Forskolin againstN. fowleri. Thymol showed the highest amoebicidal activity with IC50/24 h at 153.601 +/- 19.6 mu M. Two combinations of compounds Forskolin + Thymol and Forskolin + Borneol showed a higher effect on the viability of trophozoites as compared to compounds alone and hence showed a synergistic effect. The IC50 reported for Forskolin + Thymol was 81.30 +/- 6.86 mu M. Borneol showed maximum cysticidal activity with IC50/24 h at 192.605 +/- 3.01 mu M. Importantly, lactate dehydrogenase release testing revealed that all compounds displayed minimal cytotoxicity to human HaCaT, HeLa, and SH-SY5Y cell lines. The cytopathogenicity assay showed that Thymol and Borneol also significantly reduced the host cell cytotoxicity of pretreated amoeba toward the human HaCaT cell line. So, these terpene compounds hold potential as therapeutic agents against infections caused by N. fowleri and are potentially a step forward in drug development against this deadly pathogen as these compounds have also been reported to cross the blood-brain barrier. Therefore, an in vivo study using animal models is necessary to assess the efficacy of these compounds and the need for further research into the intranasal route of delivery for the treatment of these life-threatening infections.Öğe Potential anti-amoebic effects of synthetic 1,4-benzothiazine derivatives against Acanthamoeba castellanii(Cell Press, 2024) Alishba; Ahmed, Usman; Taha, Muhammad; Khan, Naveed Ahmed; Salar, Uzma; Khan, Khalid Mohammed; Anwar, AyazA rare but lethal central nervous system disease known as granulomatous amoebic encephalitis (GAE) and potentially blinding Acanthamoeba keratitis are diseases caused by free-living Acanthamoeba. Currently, no therapeutic agent can completely eradicate or prevent GAE. Synthetic compounds are a likely source of bioactive compounds for developing new drugs. This study synthesized seventeen 1,4-benzothiazine derivatives (I -XVII) by a base-catalyzed one-pot reaction of 2-amino thiophenol with substituted bromo acetophenones. Different spectroscopic techniques, such as EI-MS, H-1-, and C-13 NMR (only for the new compounds), were used for the structural characterization and conformation of compounds. These compounds were assessed for the first time against Acanthamoeba castellanii. All compounds showed anti-amoebic potential in vitro against A. castellanii, reducing its ability to encyst and excyst at 100 mu M. Compounds IX, X, and XVI showed the most potent activities among all derivatives and significantly reduced the viability to 5.3 x 10(4) (p < 0.0003), 2 x 10(5) (p < 0.006), and 2.4 x 10(5) (p < 0.002) cells/mL, respectively. The cytotoxicity profile revealed that these molecules showed lower to moderate cytotoxicity, i.e., 36 %, 2 %, and 21 %, respectively, against human keratinocytes in vitro. These results indicate that 1,4-benzothiazines showed potent in vitro activity against trophozoites and cysts of A. castellanii. Hence, these 1,4-benzothiazine derivatives should be considered to develop new potential therapeutic agents against Acanthamoeba infections.Öğe Self-assembled micelles loaded with itraconazole as anti-Acanthamoeba nano-formulation(Springer, 2024) Rao, Komal; Abdullah, Muhammad; Ahmed, Usman; Wehelie, Hashi Isse; Shah, Muhammad Raza; Siddiqui, Ruqaiyyah; Khan, Naveed A.Acanthamoeba castellanii are opportunistic pathogens known to cause infection of the central nervous system termed: granulomatous amoebic encephalitis, that mostly effects immunocompromised individuals, and a sight threatening keratitis, known as Acanthamoeba keratitis, which mostly affects contact lens wearers. The current treatment available is problematic, and is toxic. Herein, an amphiphilic star polymer with AB(2) miktoarms [A = hydrophobic poly(-Caprolacton) and B = hydrophilic poly (ethylene glycol)] was synthesized by ring opening polymerization and Cu-I catalyzed azide-alkyne cycloaddition. Characterization by H-1 and C-13 NMR spectroscopy, size-exclusion chromatography and fluorescence spectroscopy was accomplished. The hydrophobic drug itraconazole (ITZ) was incorporated in self-assembled micellar structure of AB(2) miktoarms through co-solvent evaporation. The properties of ITZ loaded (ITZ-PCL-PEG(2)) and blank micelles (PCL-PEG(2)) were investigated through zeta sizer, scanning electron microscopy and Fourier-transform infrared spectroscopy. Itraconazole alone (ITZ), polymer (DPB-PCL), empty polymeric micelles (PCL-PEG(2)) alone, and itraconazole loaded in polymeric micelles (ITZ-PCL-PEG(2)) were tested for anti-amoebic potential against Acanthamoeba, and the cytotoxicity on human cells were determined. The polymer was able to self-assemble in aqueous conditions and exhibited low value for critical micelle concentration (CMC) 0.05-0.06 mu g/mL. The maximum entrapment efficiency of ITZ was 68%. Of note, ITZ, DPB, PCL-PEG(2) and ITZ-PCL-PEG(2) inhibited amoebae trophozoites by 37.34%, 36.30%, 35.77%, and 68.24%, respectively, as compared to controls. Moreover, ITZ-PCL-PEG(2) revealed limited cytotoxicity against human keratinocyte cells. These results are indicative that ITZ-PCL-PEG(2) micelle show significantly better anti-amoebic effects as compared to ITZ alone and thus should be investigated further in vivo to determine its clinical potential.
