Life Sciences

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Shiv Nadar University

This work systematically describes one-step synthesis of hydrophilic functionalized superparamagnetic iron oxide nanoparticles (SPIONs) via thermolysis in presence of polyamines such as diethylene triamine (DETA), triethylene tetraamine (TETA), tetraethylene pentamine (TTEPA), or pentaethylene hexamine (PEHA) or mixture of a polyamine (TETA) and polyols such as diethylene glycol (DEG), triethylene glycol (TEG) or tetraethylene glycol (TTEG) while varying the polyamine: polyol (v/v) ratio, reaction temperature and reaction time. The saturation magnetization (Ms) values of the as-prepared polyamine (DETA/TETA/TTEPA/PEHA) coated SPIONs are determined in the range of 31.8–48.5 emu/g, which is altered in the range of 40.2–57.8 emu/g by the addition of a polyol (DEG/TEG/TTEG) to the polyamine (TETA) at different ratios. Moreover, the Ms. values are further improved to 64.6 and 66.8 emu/g at the optimized TETA:TEG (1:1) ratio by prolonging the reaction time up-to 2 h and the reaction temperature to 270 °C, respectively. In addition, the TETA-TEG coated SPIONs have displayed their average particle sizes, hydrodynamic sizes, and zeta potential (ζ) values in the range of 7–11 nm, 99–120 nm and +45 to +57 mV, respectively (indicating high water solubility). Finally, the TETA-TEG coated SPIONs with the highest Ms. and ζ values (i.e. 66.8 emu/g and +57 mV) are selected for the biological studies, where they have revealed excellent (i) cytocompatibility, and (ii) intracellular uptake in the cancer (HepG2 liver & MCF-7 breast) cells for the incubation periods of 24/48 h. Thus, the TETA-TEG coated SPIONs based aqueous ferrofluids have a great potential to be used in biomedical applications. © 2018 Elsevier B.V.

(SoNS) School of Natural Sciences

Journal of Molecular Liquids

One-step synthesis of hydrophilic functionalized and cytocompatible superparamagnetic iron oxide nanoparticles (SPIONs) based aqueous ferrofluids for biomedical applications

Zeta-toxin is a cognate toxin of epsilon antitoxin of prokaryotic Type II toxin-antitoxin system (TA) and play an important role in cell death. An orthologue of bacterial-zeta-toxin (BzT) was identified in Leishmania donovani with similar structural and functional features. Leishmania zeta-toxin (named Ld_ζ1) harboring similar UNAG and ATP-binding pockets showed UNAG kinase and ATP-binding activity. An active Ld_ζ1 was found to express in infective extracellular promastigotes stage of L. donovani and episomal overexpression of an active Ld_ζ1domain-triggered cell death. This study demonstrates the presence of prokaryotic-like-zeta-toxin in eukaryotic parasite Leishmania and its association with cell death. Conceivably, phosphorylated UNAG or analogues, the biochemical mimics of zeta-toxin function mediating cell death can act as a novel anti-leishmanial chemotherapeutics. © 2019 Federation of European Biochemical Societies

Fulltext

FEBS Letters

Identification and functional characterization of a bacterial homologue of Zeta toxin in Leishmania donovani

Lipid-based palmitoylation is a post-translation modification (PTM) which acts as a biological rheostat in life cycle progression of a deadly human malaria parasite, Plasmodium falciparum. P. falciparum palmitoylation is catalyzed by 12 putative palmitoyl acyl-transferase enzymes containing the conserved DHHC-CRD (DHHC motif within a cysteine-rich domain) which can serve as a druggable target. However, the paucity of high-throughput assays has impeded the design of drugs targeting palmitoylation. We have developed a novel strategy which involves engineering of Escherichia coli, a PTM-null system, to enforce ectopic expression of palmitoyl acyl-transferase in order to study Plasmodium-specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic E. coli strains expressing Plasmodium-specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl-transferase activity via acyl-biotin exchange (ABE) and clickable chemistry methods. E. coli proteome was found to be palmitoylated in PfDHHC-expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of E. coli proteins. Upon treatment with generic inhibitor 2-bromopalmitate (2-BMP), a predominant reduction in palmitic acid incorporation is detected. Overall, these findings suggest that synthetic E. coli strains expressing PfDHHCs can enforce global palmitoylation in the E. coli proteome. Interestingly, this finding was corroborated by our in silico palmitoylome profiling, which revealed that out of the total E. coli proteome, 108 proteins were predicted to be palmitoylated as represented by the presence of three cysteine consensus motifs (cluster type I, II, III). In summary, our study reports a proof of concept for screening of chemotherapeutics targeting the palmitoylation machinery using a high-throughput screening platform. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

World University root node

FEBS Open Bio

Plasmodium palmitoylation machinery engineered in E. coli for high-throughput screening of palmitoyl acyl-transferase inhibitors

Novel experimental and sequencing techniques have led to an exponential explosion and spiraling of data in viral genomics. To analyse such data, rapidly gain information, and transform this information to knowledge, interdisciplinary approaches involving several different types of expertise are necessary. Machine learning has been in the forefront of providing models with increasing accuracy due to development of newer paradigms with strong fundamental bases. Support Vector Machines (SVM) is one such robust tool, based rigorously on statistical learning theory. SVM provides very high quality and robust solutions to classification and regression problems. Several studies in virology employ high performance tools including SVM for identification of potentially important gene and protein functions. This is mainly due to the highly beneficial aspects of SVM. In this chapter we briefly provide lucid and easy to understand details of SVM algorithms along with applications in virology. © Springer Nature Switzerland AG 2019. All rights reserved.

Global Virology III: Virology in the 21st Century

Application of support vector machines in viral biology

Malaria, one of the most severe global diseases, infects nearly 300 million people causing death of about a million population annually. Herein we have reported design, synthesis and biological evaluation of potent antimalarial compounds that target melatonin hormone as a potential pathway for the inhibition of the parasite proliferation. The molecular design is based on melatonin and indole based synthetic and natural antimalarial agents. The library of compounds was accessed via an iodine catalyzed one pot organocatalytic ring opening of 1-aryltetrahydro-β-carbolines followed by in situ imination of the resulting C2-aroyl intermediates. Inhibition of parasite growth progression (3D7 and chloroquine resistant RKL9 strain) in the presence of the tested compounds indicated that few of the compounds substantially inhibited the parasite survival and the most potent compound 2j blocked the parasite growth at the trophozoite stage. Compound 2j also disrupted the melatonin induced synchronization of the parasite culture in vitro. The active compounds were screened against melatonin receptor MT1 to demonstrate substantial binding. © 2019 Elsevier Masson SAS

Chemistry

European Journal of Medicinal Chemistry

Indole based antimalarial compounds targeting the melatonin pathway: Their design, synthesis and biological evaluation

Synthetic organic molecules, which can selectively convert excess intracellular copper (Cu) ions to nanozymes with an ability to protect cells from oxidative stress, are highly significant in developing therapeutic agents against Cu-related disorder like Wilson's disease. Here, we report 1,3-bis(2-hydroxyethyl)-1H-benzoimidazole-2-selenone (1), which shows a remarkable ability to remove Cu ion from glutathione, a major cytosolic Cu-binding ligand, and thereafter converts it into copper selenide (CuSe) nanozyme that exhibits remarkable glutathione peroxidase-like activity, at cellular level of H 2 O 2 concentration, with excellent cytoprotective effect against oxidative stress in hepatocyte. Cu-driven deselenization of 1, under physiologically relevant conditions, occurred in two steps. The activation of C=Se bond by metal ion is the crucial first step, followed by cleavage of the metal-activated C=Se bond, initiated by the OH group of N-(CH 2 ) 2 OH substituent through neighboring group participation (deselenization step), resulted in the controlled synthesis of various types of Cu 2-x Se nanocrystals (NCs) (nanodisks, nanocubes, and nanosheets) and tetragonal Cu 3 Se 2 NCs, depending upon the oxidation state of the Cu ion used to activate the C=Se bond. Deselenization of 1 is highly metal-selective. Except Cu, other essential metal ions, including Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , or Zn 2+ , failed to produce metal selenide under identical reaction conditions. Moreover, no significant change in the expression level of Cu-metabolism-related genes, including metallothioneines MT1A, is observed in liver cells co-treated with Cu and 1, as opposed to the large increase in the concentrations of these genes observed in cells treated with Cu alone, suggesting the participation of 1 in Cu homeostasis in hepatocyte. © 2019 American Chemical Society.

ACS Applied Materials and Interfaces

Copper-Driven Deselenization: A Strategy for Selective Conversion of Copper Ion to Nanozyme and Its Implication for Copper-Related Disorders

Clostridium perfringens epsilon toxin (Etx) is categorized as the third most lethal bioterrorism agent by the Centers for Disease Control and Prevention (CDC), with no therapeutic counter measures available for humans. Here, we have developed a high-affinity inhibitory compound by synthesizing and evaluating the structure activity relationship (SAR) of a library of diverse glycosides (numbered 1-12). SAR of glycoside-Etx heptamers revealed exceptionally strong H-bond interactions of glycoside-4 with a druggable pocket in the oligomerization and β-hairpin region of Etx. Analysis of its structure suggested that glycoside-4 might self-aggregate to form a robust micelle-like supra-molecular complex due to its linear side-chain architecture, which was authenticated by fluorescence spectroscopy. Further, this micelle hinders the Etx monomer-monomer interaction required for oligomerization, validated by both surface plasmon resonance (SPR) and immunoblotting. This phenomenon in turn leads to blockage of pore formation. Downstream evaluation revealed that glycoside-4 effectively blocked cell death of Etx-treated cultured primary cells and maintained cellular homeostasis via disrupting oligomerization, blocking pore formation, restoring calcium homeostasis, stabilizing the mitochondrial membrane and impairing high mobility group box 1 (HMGB1) translocation from nucleus to cytoplasm. Furthermore, a single dosage of glycoside-4 protected the Etx-challenged mice and restored normal function to multiple organs. This work reports for the first time a potent, nontoxic glycoside with strong ability to occlude toxin lethality, representing it as a bio-arm therapeutic against Etx-based biological threat. © 2019. Published by The Company of Biologists Ltd

DMM Disease Models and Mechanisms

A small bioactive glycoside inhibits epsilon toxin and prevents cell death

In pre-clinical models, co-existence of Human Epidermal Growth Factor Receptor-2 (HER2)-amplification and PI3K catalytic subunit (PIK3CA) mutations results in aggressive, anti-HER2 therapy-resistant breast tumors. This is not always reflected in clinical setting. We speculated that the complex interaction between the HER2 and PIK3CA oncogenes is responsible for such inconsistency. We performed series of biochemical, molecular and cellular assays on genetically engineered isogenic mammary epithelial cell lines and breast cancer cells expressing both oncogenes. In vitro observations were validated in xenografts models. We showed that H1047R, one of the most common PIK3CA mutations, is responsible for endowing a senescence-like state in mammary epithelial cells overexpressing HER2. Instead of imposing a permanent growth arrest characteristic of oncogene-induced senescence, the proteome secreted by the mutant cells promotes stem cell enrichment, angiogenesis, epithelial-to-mesenchymal transition, altered immune surveillance and acute vulnerability toward HSP90 inhibition. We inferred that the pleiotropism, as observed here, conferred by the mutated oncogene, depending on the host microenvironment, contributes to conflicting pre-clinical and clinical characteristics of HER2+, mutated PIK3CA-bearing tumor cells. We also came up with a plausible model for evolution of breast tumors from mammary epithelial cells harboring these two molecular lesions. © 2019 The Author(s). Published by Oxford University Press. All rights reserved.

Carcinogenesis

The H1047R PIK3CA oncogene induces a senescence-like state, pleiotropy and acute HSP90 dependency in HER2+ mammary epithelial cells

Malaria, caused by protozoan of genus Plasmodium, remains one of the highest mortality infectious diseases. Malaria parasites have a complex life cycle, easily adapt to their host’s immune system and have evolved with an arsenal of unique proteases which play crucial roles in proliferation and survival within the host cells. Owing to the existing knowledge of enzymatic mechanisms, 3D structures and active sites of proteases, they have been proven to be opportune for target based drug development. Here, we discuss in depth the crucial roles of essential proteases in Plasmodium life cycle and particularly focus on highlighting the atypical “structural signatures” of key parasite proteases which have been exploited for drug development. These features, on one hand aid parasites pathogenicity while on the other hand could be effective in designing targeted and very specific inhibitors for counteracting them. We conclude that Plasmodium proteases are suitable as multistage targets for designing novel drugs with new modes of action to combat malaria. Copyright © 2019 Mishra, Singh and Singh. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Frontiers in Microbiology

Structural insights into key plasmodium proteases as therapeutic drug targets

Successful pathogenesis is a cumulative effect of the virulence factors of a pathogen and its capability to efficiently utilize the available nutrients from the host. Streptococcus pneumoniae, a Gram-positive opportunistic pathogen, may either reside asymptomatically as a nasopharyngeal commensal inside the human host or cause lethal diseases, including pneumonia, meningitis and sepsis. S. pneumoniae is known to acquire methionine (Met) from its host through a Met importer. Here, the crystal structure of the substrate-binding protein (SBP; SP_0149) of an ABC importer with Met bound is reported at a resolution of 1.95 {\AA}. The three-dimensional structure of SBP shows that it is composed of two distinct domains, each consisting of a mixed $\beta$-sheet flanked by helices. The substrate, Met, is bound in the central part of the interface between the two domains. The overall structure of SP_0149 resembles those of SBPs from other reported bacterial Met and Gly-Met dipeptide transporters. However, a detailed analysis of these structures shows notable variations in the amino-acid composition of the substrate-binding pockets of the SP_0149-Met and GmpC-Gly-Met structures. In particular, SP_0149 harbors Thr212 and Tyr114, whereas the corresponding residues in GmpC are Gly and Val. This difference is likely to be the underlying basis for their differential substrate specificity. In summary, the structure of the SP_0149-Met complex provides insights into the transport function of SP_0149 and its interactions with methionine. It opens up avenues for the rational design of inhibitors of SP_0149 through a structure-mediated approach.

Acta Crystallographica Section F: Structural Biology Communications

Structural insights into the substrate specificity of SP_0149, the substrate-binding protein of a methionine ABC transporter from Streptococcus pneumoniae