ABSTRACT
Leishmania encode six paralogs of the cap-binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap-binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C-terminal extension was identified through structure prediction and sequence alignment. m7 GTP-binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C-terminal extension revealed that this extension functions as a regulatory gate, modulating the cap-binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C-terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem-loop structure in the 3' UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis, a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S-phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S-phase-abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.
Subject(s)
Leishmania , RNA Cap-Binding Proteins/metabolism , Histones/metabolism , Proteomics , RNA, Messenger/metabolism , Cell Cycle , Protein BindingABSTRACT
Copper(I)-catalyzed 1,3-dipolar cycloaddition between organic azides and terminal alkynes, commonly known as CuAAC or click chemistry, has been identified as one of the most successful, versatile, reliable, and modular strategies for the rapid and regioselective construction of 1,4-disubstituted 1,2,3-triazoles as diversely functionalized molecules. Carbohydrates, an integral part of living cells, have several fascinating features, including their structural diversity, biocompatibility, bioavailability, hydrophilicity, and superior ADME properties with minimal toxicity, which support increased demand to explore them as versatile scaffolds for easy access to diverse glycohybrids and well-defined glycoconjugates for complete chemical, biochemical, and pharmacological investigations. This review highlights the successful development of CuAAC or click chemistry in emerging areas of glycoscience, including the synthesis of triazole appended carbohydrate-containing molecular architectures (mainly glycohybrids, glycoconjugates, glycopolymers, glycopeptides, glycoproteins, glycolipids, glycoclusters, and glycodendrimers through regioselective triazole forming modular and bio-orthogonal coupling protocols). It discusses the widespread applications of these glycoproducts as enzyme inhibitors in drug discovery and development, sensing, gelation, chelation, glycosylation, and catalysis. This review also covers the impact of click chemistry and provides future perspectives on its role in various emerging disciplines of science and technology.
Subject(s)
Click Chemistry , Copper/chemistry , Glycoconjugates/chemistry , Animals , Catalysis , Humans , Triazoles/chemistryABSTRACT
To develop a better chemotherapeutically potential candidate for lung cancer treatment and cure with repurposed motifs, quinine has been linked with biocompatible CuAAC-inspired regioselective 1,2,3-triazole linker and a series of ten novel 1,2,3-triazolyl-9-quinine conjugates have been developed by utilizing click conjugation of glycosyl ether alkynes with 9-epi-9-azido-9-deoxy-quinine under standard click conditions. In parallel, the docking study indicated that the resulting conjugates have an overall appreciable interaction with ALK-5 macromolecules. Moreover, the mannose-triazolyl conjugate exhibited the highest binding interactions of -7.6â kcal/mol with H-bond interaction with the targeted macromolecular system and indicate the hope for future trials for anti-lung cancer candidates.
Subject(s)
Quinine , Quinine/pharmacology , Molecular Docking SimulationABSTRACT
BACKGROUND: The biotherapeutic asparaginase is a cornerstone of therapy in acute lymphoblastic leukaemia (ALL). With limited access to the original native Escherichia coli-derived asparaginase (EcASNase), a variety of EcASNase biogenerics are used in low-middle-income countries (LMICs). The variable quality of these biogenerics potentially influences clinical outcomes. PROCEDURE: Seven biogeneric EcASNases (P1-P7) marketed widely in India were evaluated, with P2 as an exemplar for in vivo monitoring. Therapeutic activity of P2 (10,000 IU/m2 /dose, intramuscular, every 72 hours) was monitored during induction therapy, and drug-related toxicities recorded. Molecular identity, purity and in vitro drug activity of seven biogenerics were characterised using multimodal analyses, and findings compared with reference EcASNase (R). RESULTS: In patients (N = 62) receiving P2, subtherapeutic asparaginase activity (<100 U/L) was observed in 66% (46/70) of trough timepoints (72 hours postdose) during induction. Twelve patients (19%), 11 with high-risk ALL, developed hypersensitivity. Isoforms of EcASNase were identified in all seven biogenerics. All generic products contained impurities with batch-to-batch variability. These included high levels of protein aggregates and host cell protein contamination. In vitro assays of EcASNase activity and leukaemia cell line cytotoxicity were not discriminatory. CONCLUSIONS: Our findings confirm widespread concerns over the unsatisfactory quality and therapeutic activity of native EcASNase biogenerics marketed in LMICs. Appropriate use of these products requires monitored studies to identify clinical suitability and determine appropriate dosing and schedule. For large parts of the world, assured access to high-quality asparaginases remains an unmet therapeutic need.
Subject(s)
Antineoplastic Agents , Asparaginase , Biological Products/therapeutic use , Drugs, Generic/therapeutic use , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Antineoplastic Agents/therapeutic use , Asparaginase/therapeutic use , Escherichia coli/enzymology , Humans , India , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Therapeutic EquivalencyABSTRACT
The recent emergence of hypervirulent clinical variants of Klebsiella pneumoniae (hvKP) causing community-acquired, invasive, metastatic, life-threatening infections of lungs, pleura, prostate, bones, joints, kidneys, spleen, muscles, soft-tissues, skin, eyes, central nervous system (CNS) including extrahepatic abscesses, and primary bacteremia even in healthy individuals has posed stern challenges before the existing treatment modalities. There is therefore an urgent need to look for specific and effective therapeutic alternatives against the said bacterial infection or recurrence. A new type of MoS2-modified curcumin nanostructure has been developed and evaluated as a potential alternative for the treatment of multidrug-resistant isolates. The curcumin quantum particles have been fabricated with MoS2 via a seed-mediated hydrothermal method, and the resulting MoS2-modified curcumin nanostructures (MQCs) have been subsequently tested for their antibacterial and antibiofilm properties against hypervirulent multidrug-resistant Klebsiella pneumoniae isolates. In the present study, we found MQCs inhibiting the bacterial growth at a minimal concentration of 0.0156 µg/mL, while complete inhibition of bacterial growth was evinced at concentration 0.125 µg/mL. Besides, we also investigated their biocompatibility both in vitro and in vivo. MQCs were found to be nontoxic to the SiHa cells at a dose as high as 1024 µg/mL on the basis of the tested adhesion, spreading of the cells, and also on the various serological, biochemical, and histological investigations of the vital organs and blood of the Charles Foster Rat. These results suggest that MQCs have potent antimicrobial activities against hvKP and other drug resistant isolates and therefore may be used as broad spectrum antibacterial and antibiofilm agents.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Curcumin/pharmacology , Disulfides/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Klebsiella pneumoniae/drug effects , Molybdenum/pharmacology , Nanostructures/chemistry , Theranostic Nanomedicine , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Curcumin/chemical synthesis , Curcumin/chemistry , Disulfides/chemistry , Microbial Sensitivity Tests , Molybdenum/chemistryABSTRACT
Background: Most patients with transient ischaemic attack (TIA) present to their GP. Early identification and treatment reduces the risk of subsequent stroke and consequent disability and mortality. Objective: To explore GPs' views on the diagnosis and immediate management of suspected TIA, and the potential utility of a diagnostic tool. Methods: This is a qualitative interview study based in Leicestershire, UK. A purposive sample of 10 GPs participated in 30-minute semi-structured telephone interviews. Data were analysed thematically. Results: GPs reported that TIA was more likely to be suspected when patients were more obvious candidates for TIA based on their history, characteristics and symptom presentation. Referrals were in part a strategy to manage risk under conditions of uncertainty and to seek reassurance. GPs valued using a TIA risk stratification tool but felt this did not inform their diagnostic decision making. A diagnostic tool for TIA in primary care was seen to have potential to improve the decision-making process about diagnosis and management and enhance confidence of GPs, particularly in ruling out TIAs. GPs saw benefits of using hard thresholds, but remained concerned about missing TIAs and saw a tool as an adjunct to clinical judgement. Conclusions: GPs weigh up the likelihood of TIA in the context of assessments of candidacy and diverse, often vague, symptoms. A diagnostic tool could support GPs in this process and help reduce reliance on referrals to TIA clinics for reassurance, provided the tool was designed to support decision making in cases of less 'typical' presentations.
Subject(s)
General Practitioners/psychology , Ischemic Attack, Transient/diagnostic imaging , Ischemic Attack, Transient/therapy , Stroke/prevention & control , Uncertainty , Female , Humans , Male , Middle Aged , Qualitative Research , Referral and Consultation , Surveys and QuestionnairesABSTRACT
To achieve better-repurposed motifs, saccharin has been merged with biocompatible sugar molecules via a 1,2,3-triazole linker, and ten novel 1,2,3-triazole-appended saccharin glycoconjugates were developed in good yield by utilizing modular CuAAC click as regioselective triazole forming tool. The docking study indicated that the resulting hybrid molecules have an overall substantial interaction with the CAXII macromolecule. Moreover, the galactose triazolyl saccharin analogue 3h has a binding energy of -8.5 kcal/mol with 5 H-bonds, and xylosyl 1,2,3-triazolyl saccharin analogue 3d has a binding energy of -8.2 kcal/mol with 6 H-bond interactions and have exhibited the highest binding interaction with the macromolecule system.
Subject(s)
Click Chemistry , Saccharin , Click Chemistry/methods , Glycoconjugates/chemistry , Triazoles/chemistry , Molecular Docking SimulationABSTRACT
Translation of most cellular mRNAs in eukaryotes proceeds through a cap-dependent pathway, whereby the cap-binding complex, eIF4F, anchors the pre-initiation complex at the 5' end of mRNAs driving translation initiation. The genome of Leishmania encodes a large repertoire of cap-binding complexes that fulfill a variety of functions possibly involved in survival along the life cycle. However, most of these complexes function in the promastigote life form that resides in the sand fly vector and decrease their activity in amastigotes, the mammalian life form. Here we examined the possibility that LeishIF3d drives translation in Leishmania using alternative pathways. We describe a non-canonical cap-binding activity of LeishIF3d and examine its potential role in driving translation. LeishIF3d is required for translation, as reducing its expression by a hemizygous deletion reduces the translation activity of the LeishIF3d(+/-) mutant cells. Proteomic analysis of the mutant cells highlights the reduced expression of flagellar and cytoskeletal proteins, as reflected in the morphological changes observed in the mutant cells. Targeted mutations in two predicted alpha helices diminish the cap-binding activity of LeishIF3d. Overall, LeishIF3d could serve as a driving force for alternative translation pathways, although it does not seem to offer an alternative pathway for translation in amastigotes.
ABSTRACT
In nature, almost all cells are covered with a complex array of glycan chain namely sialic acids or nuraminic acids, a negatively charged nine carbon sugars which is considered for their great therapeutic importance since long back. Owing to its presence at the terminal end of lipid bilayer (commonly known as terminal sugars), the well-defined sialosides or sialoconjugates have served pivotal role on the cell surfaces and thus, the sialic acid-containing glycans can modulate and mediate a number of imperative cellular interactions. Understanding of the sialo-protein interaction and their roles in vertebrates in regard of normal physiology, pathological variance, and evolution has indeed a noteworthy journey in medicine. In this tutorial review, we present a concise overview about the structure, linkages in chemical diversity, biological significance followed by chemical and enzymatic modification/synthesis of sialic acid containing glycans. A more focus is attempted about the recent advances, opportunity, and more over growing impact of sialosides and sialoconjugates in future drug discovery and development.
Subject(s)
N-Acetylneuraminic Acid , Sialic Acids , Animals , N-Acetylneuraminic Acid/chemistry , Sialic Acids/chemistry , Polysaccharides/chemistry , Sialyltransferases/metabolism , SugarsABSTRACT
To imbibe the aim of synthesizing water-soluble and biocompatible motif, a click-inspired piperazine glycoconjugate has been devised up. In this report, we present a focused approach to design and synthesis of versatile sugar-appended triazoles through 'Click Chemistry' along with their pharmacological studies on cyclin-dependent kinases (CDKs) and cell cytotoxicity on cancer cells using in silico and in vitro approaches, respectively. The study has inclusively recognized the galactose- and mannose-derived piperazine conjugates as the promising motifs. The findings suggested that the galactosyl bis-triazolyl piperazine analogue 10b is the most CDK interactive derivative and also possess significant anticancer activity.
Subject(s)
Antineoplastic Agents , Sugars , Piperazine/pharmacology , Click Chemistry , Glycoconjugates , Galactose , Antineoplastic Agents/pharmacologyABSTRACT
Among all the malaria parasites, P. falciparum is the most predominant species which has developed drug resistance against most of the commercial anti-malarial drugs. Thus, finding a new molecule for the inhibition of enzymes of P. falciparum is the pharmacological challenge in present era. Herein, ten novel molecules have been designed with an amalgamation of cinchonidine, carbohydrate moiety and triazole ring by utilizing copper-catalyzed click reaction of cinchonidine-derived azide and clickable glycosyl alkynes. The molecular docking of developed molecules showed promising results for plasmepsin inhibition in the form of effective binding with target proteins.
Subject(s)
Antimalarials/chemical synthesis , Aspartic Acid Endopeptidases/antagonists & inhibitors , Cinchona Alkaloids/chemistry , Plasmodium falciparum/drug effects , Protease Inhibitors/chemical synthesis , Protozoan Proteins/antagonists & inhibitors , Antimalarials/chemistry , Antimalarials/pharmacology , Aspartic Acid Endopeptidases/chemistry , Catalysis , Cinchona Alkaloids/chemical synthesis , Cinchona Alkaloids/pharmacology , Click Chemistry , Copper/chemistry , Drug Design , Humans , Malaria, Falciparum/parasitology , Molecular Docking Simulation , Molecular Structure , Plasmodium falciparum/enzymology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protozoan Proteins/chemistry , Triazoles/chemistryABSTRACT
Epstein-Barr virus (EBV) oncoprotein EBNA3C is indispensable for primary B-cell transformation and maintenance of lymphoblastoid cells outgrowth. EBNA3C usurps two putative cellular pathways-cell-cycle and apoptosis, essentially through modulating ubiquitin-mediated protein-degradation or gene transcription. In cancer cells, these two pathways are interconnected with autophagy,-a survival-promoting catabolic network in which cytoplasmic material including mis/un-folded protein aggregates and damaged organelles along with intracellular pathogens are degraded and recycled in lysosomal compartments. Studies have shown that tumor viruses including EBV can manipulate autophagy as a survival strategy. Here, we demonstrate that EBNA3C elevates autophagy, which serves as a prerequisite for apoptotic inhibition and maintenance of cell growth. Using PCR based micro-array we show that EBNA3C globally accelerates autophagy gene transcription under growth limiting conditions. Reanalyzing the ENCODE ChIP-sequencing data (GSE52632 and GSE26386) followed by ChIP-PCR demonstrate that EBNA3C recruits several histone activation epigenetic marks (H3K4me1, H3K4me3, H3K9ac, and H3K27ac) for transcriptional activation of autophagy genes, notably ATG3, ATG5, and ATG7 responsible for autophagosome formation. Moreover, under growth limiting conditions EBNA3C further stimulates the autophagic response through upregulation of a number of tumor suppressor genes, notably cyclin-dependent kinase inhibitors-CDKN1B (p27Kip1) and CDKN2A (p16INK4a) and autophagy mediated cell-death modulators-DRAM1 and DAPK1. Together our data highlight a new role of an essential EBV oncoprotein in regulating autophagy cascade as a survival mechanism and offer novel-targets for potential therapeutic expansion against EBV induced B-cell lymphomas.
Subject(s)
Autophagy/genetics , B-Lymphocytes/pathology , Epigenesis, Genetic , Herpesvirus 4, Human/metabolism , Transcription, Genetic , Viral Proteins/metabolism , Apoptosis/genetics , Autophagy-Related Protein 5/metabolism , B-Lymphocytes/metabolism , Cell Cycle/genetics , Cell Proliferation/genetics , Cell Survival/genetics , Cytoprotection , HEK293 Cells , Histones/metabolism , Humans , Models, BiologicalABSTRACT
INTRODUCTION Many patients who suffer a transient ischaemic attack (TIA) present to their general practitioner (GP). Early identification and treatment reduces the risk of subsequent stroke, disability and mortality. AIM To review the accuracy of TIA diagnosis in primary care, immediate management and interventions to assist GPs with the condition. METHODS This study included the search of Medline, Embase, Web of Science and Scopus databases (1995-2015). Relevant titles and abstracts were obtained using structured criteria (diagnosis, immediate management or intervention of TIAs in primary care), with full review and data extraction for eligible publications. RESULTS Most studies found limitations in GPs' knowledge and ability to diagnose TIAs to varying extent over time and between countries. GPs tended to over-interpret non-specific symptoms (e.g. isolated vertigo) when considering a TIA diagnosis. Reported referral behaviour varied between countries, with some favouring admission and others preferring outpatient management. Consistent under-referral and under-use of effective medication was reported. However, GPs may refer some patients to exclude rather than confirm a final diagnosis. This, alongside evidence of under-referral, suggests the need for education and decision support tools to enhance referral patterns. Intervention studies suggested that electronic decision support may increase referrals and timely management. CONCLUSION This review revealed deficiencies in knowledge and clinical practice, and identified potential avenues to addressing these. Issues for future research were also identified.
Subject(s)
Ischemic Attack, Transient/diagnosis , Ischemic Attack, Transient/drug therapy , Primary Health Care , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle AgedABSTRACT
Epstein-Barr virus (EBV) is highly ubiquitous in human population and establishes a lifelong asymptomatic infection within the infected host unless the immune system is compromised. Following initial infection in the oropharyngeal epithelial cells, EBV primarily infects naive B-lymphocytes and develops a number of B-cell lymphomas particularly in immune-deficient individuals. In vitro, EBV can also infect and subsequently transform quiescent B-lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs) resembling EBV-induced lymphoproliferative disorders in which a subset of latent transcripts are detected. Genetic studies revealed that EBNA-3 family comprising of three adjacent genes in the viral genome-EBNA-3A and -3C, but not -3B, are critical for B-cell transformation. Nevertheless, all three proteins appear to significantly contribute to maintain the overall proliferation and viability of transformed cells, suggesting a critical role in lymphoma development. Apart from functioning as important viral transcriptional regulators, EBNA-3 proteins associate with many cellular proteins in different signaling networks, providing a suitable platform for lifelong survival of the virus and concurrent lymphoma development in the infected host. The chapter describes the function of each these EBV nuclear antigen 3 proteins employed by the virus as a means to understand viral pathogenesis of several EBV-associated B-cell malignancies.