ABSTRACT
Sibiriline is a novel drug inhibiting receptor-interacting protein 1 kinase (RIPK1) and necroptosis, a regulated form of cell death involved in several disease models. In this study, we aimed to investigate the metabolic fate of sibiriline in a cross-sectional manner using an in silico prediction, coupled with in vitro and in vivo experiments. In silico predictions were performed using GLORYx and Biotransformer 3.0 freeware; in vitro incubation was performed on differentiated human HepaRG cells, and in vivo experiments including a pharmacokinetic study were performed on mice treated with sibiriline. HepaRG culture supernatants and mice plasma samples were analyzed with ultra-high-performance liquid chromatography, coupled with tandem mass spectrometry (LC-HRMS/MS). The molecular networking bioinformatics tool applied to LC-HRMS/MS data allowed us to visualize the sibiriline metabolism kinetics. Overall, 14 metabolites, mostly produced by Phase II transformations (glucuronidation and sulfation) were identified. These data provide initial reassurance regarding the toxicology of this new RIPK1 inhibitor, although further studies are required.
ABSTRACT
Nigratine (also known as 6E11), a flavanone derivative of a plant natural product, was characterized as highly specific non-ATP competitive inhibitor of RIPK1 kinase, one of the key components of necroptotic cell death signaling. We show here that nigratine inhibited both necroptosis (induced by Tumor Necrosis Factor-α) and ferroptosis (induced by the small molecules glutamate, erastin, RSL3 or cumene hydroperoxide) with EC50 in the µM range. Taken together, our data showed that nigratine is a dual inhibitor of necroptosis and ferroptosis cell death pathways. These findings open potential new therapeutic avenues for treating complex necrosis-related diseases.
Subject(s)
Ferroptosis , Apoptosis , Cell Death/physiology , Humans , Necroptosis , Necrosis , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.
Subject(s)
Acacia/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Triterpenes/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Apoptosis/genetics , Binding Sites , Casein Kinase 1 epsilon/antagonists & inhibitors , Casein Kinase 1 epsilon/genetics , Casein Kinase 1 epsilon/metabolism , Cell Proliferation/drug effects , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Humans , Janus Kinase 3/antagonists & inhibitors , Janus Kinase 3/genetics , Janus Kinase 3/metabolism , K562 Cells , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Models, Molecular , NIMA-Related Kinases/antagonists & inhibitors , NIMA-Related Kinases/genetics , NIMA-Related Kinases/metabolism , Plant Bark/chemistry , Plant Extracts/chemistry , Protein Binding , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/isolation & purification , Proto-Oncogene Proteins c-abl/chemistry , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Signal Transduction , Triterpenes/chemistry , Triterpenes/isolation & purification , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
With the aim to develop new chemical tools based on simplified natural metabolites to help deciphering the molecular mechanism of necroptosis, simplified benzazole fragments including 2-aminobenzimidazole and the 2-aminobenzothiazole analogs were prepared during the synthesis of the marine benzosceptrin B. Conpounds inhibiting the RIPK1 protein kinase were discovered. A library of 54 synthetic analogs were prepared and evaluated through a phenotypic screen using the inhibition of the necrotic cell death induced by TNF-α in human Jurkat T cells deficient for the FADD protein. This article reports the design, synthesis and biological evaluation of a series of 2-aminobenzazoles on the necroptotic cell death through the inhibition of RIPK1 protein kinase. The 2-aminobenzimidazole and 2-aminobenzothiazole platforms presented herein can serve as novel chemical tools to study the molecular regulation of necroptosis and further develop lead drug candidates for chronic pathologies involving necroptosis.
Subject(s)
Imidazoles/pharmacology , Necroptosis/drug effects , Protein Kinase Inhibitors/pharmacology , Pyrroles/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Binding Sites , Drug Design , Fas-Associated Death Domain Protein/deficiency , Humans , Imidazoles/chemical synthesis , Imidazoles/metabolism , Jurkat Cells , Molecular Docking Simulation , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Pyrroles/chemical synthesis , Pyrroles/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/chemistry , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Structure-Activity RelationshipABSTRACT
Regulated cell death (RCD) results from the activation of one or more signal transduction modules both in physiological or pathological conditions. It is now established that RCD is involved in numerous human diseases, including cancer. As regulated cell death processes can be modulated by pharmacological tools, the research reported here aims to characterize new marine compounds acting as RCD modulators. Protein kinases (PKs) are key signaling actors in various RCDs notably through the control of either mitosis (e.g., the PKs Aurora A and B) or necroptosis (e.g., RIPK1 and RIPK3). From the primary screening of 27 various extracts of marine organisms collected in the Mediterranean Sea, an extract and subsequently a purified high molecular weight compound dubbed P3, were isolated from the marine sponge Crambe tailliezi and characterized as a selective inhibitor of PKs Aurora A and B. Furthermore, P3 was shown to induce apoptosis and to decrease proliferation and mitotic index of human osteosarcoma U-2 OS cells.
Subject(s)
Biological Products/pharmacology , Crambe Sponge/chemistry , Crambe Sponge/metabolism , Cytotoxins/pharmacology , Animals , Apoptosis/drug effects , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , HEK293 Cells , Hep G2 Cells , Humans , MCF-7 Cells , Mediterranean Sea , Molecular Weight , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Signal Transduction/drug effectsABSTRACT
The marine α-pyrone macrolide neurymenolide A was previously isolated from the Fijian red macroalga, Neurymenia fraxinifolia, and characterized as an antibacterial agent against antibiotic-resistant strains that also exhibited moderate cytotoxicity in vitro against cancer cell lines. This compound was also shown to exhibit allelopathic effects on Scleractinian corals. However, to date no mechanism of action has been described in the literature. The present study showed, for the first time, the isolation of neurymenolide A from the New Caledonian Rhodophyta, Phacelocarpus neurymenioides. We confirmed the compound's moderate cytotoxicity in vitro against several human cell lines, including solid and hematological malignancies. Furthermore, we combined fluorescence microscopy and flow cytometry to demonstrate that treatment of U-2 OS osteosarcoma human cells with neurymenolide A could block cell division in prometaphase by inhibiting the correct formation of the mitotic spindle, which induced a mitotic catastrophe that led to necrosis and apoptosis. Absolute configuration of the stereogenic center C-17 of neurymenolide A was deduced by comparison of the experimental and theoretical circular dichroism spectra. Since the total synthesis of this compound has already been described, our findings open new avenues in cancer treatment for this class of marine molecules, including a new source for the natural product.
Subject(s)
Macrolides/chemistry , Macrolides/pharmacology , Pyrones/chemistry , Pyrones/pharmacology , Rhodophyta/chemistry , Spindle Apparatus/drug effects , Apoptosis/drug effects , Bone Neoplasms/drug therapy , Bone Neoplasms/pathology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Hep G2 Cells , Humans , K562 Cells , MCF-7 Cells , Microtubules/pathology , Mitosis/drug effects , Necrosis/drug therapy , Osteosarcoma/drug therapy , Osteosarcoma/pathologyABSTRACT
Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.
Subject(s)
Casein Kinase 1 epsilon/antagonists & inhibitors , Casein Kinase Ialpha/antagonists & inhibitors , Kidney/drug effects , Polycystic Kidney Diseases/prevention & control , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyridines/pharmacology , Roscovitine/pharmacology , Animals , Casein Kinase 1 epsilon/genetics , Casein Kinase 1 epsilon/metabolism , Casein Kinase Ialpha/genetics , Casein Kinase Ialpha/metabolism , Catalysis , Chromatography, Affinity/methods , Disease Models, Animal , Humans , Kidney/enzymology , Kidney/pathology , Mice, Transgenic , Polycystic Kidney Diseases/enzymology , Polycystic Kidney Diseases/genetics , Polycystic Kidney Diseases/pathology , Protein Binding , Protein Kinase Inhibitors/metabolism , Purines/metabolism , Pyridines/metabolism , Roscovitine/metabolism , Signal Transduction/drug effectsABSTRACT
Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis.
Subject(s)
Alkaloids/pharmacology , Hepatitis, Animal/prevention & control , Immunologic Factors/pharmacology , Protein Kinase Inhibitors/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Spiro Compounds/pharmacology , Alkaloids/chemistry , Animals , Apoptosis/drug effects , Apoptosis/genetics , Caspase 3/genetics , Caspase 3/immunology , Cell Line, Transformed , Concanavalin A , Cycloheximide/pharmacology , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation , HT29 Cells , Hepatitis, Animal/chemically induced , Hepatitis, Animal/genetics , Hepatitis, Animal/immunology , Humans , Imidazoles/pharmacology , Immunologic Factors/chemistry , Indoles/pharmacology , Jurkat Cells , Male , Mice , Molecular Docking Simulation , Necrosis/chemically induced , Necrosis/genetics , Necrosis/immunology , Protein Kinase Inhibitors/chemistry , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/immunology , Signal Transduction , Spiro Compounds/chemistry , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
We report here the synthesis, the biological evaluation and the molecular modeling studies of new imidazo[1,2-a]pyridines derivatives designed as potent kinase inhibitors. This collection was obtained from 2-aminopyridines and 2-bromoacetophenone which afforded final compound in only one step. The bioactivity of this family of new compounds was tested using protein kinase and ATP competition assays. The structure-activity relationship (SAR) revealed that six compounds inhibit DYRK1A and CLK1 at a micromolar range. Docking studies provided possible explanations that correlate with the SAR data. The most active compound 4c inhibits CLK1 (IC50 of 0.7 µM) and DYRK1A (IC50 of 2.6 µM).
Subject(s)
Models, Molecular , Protein Kinase Inhibitors/chemical synthesis , Pyridines/pharmacology , Humans , Molecular Docking Simulation , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyridines/chemical synthesis , Pyridines/chemistry , Structure-Activity Relationship , Dyrk KinasesABSTRACT
Using a structure-based design approach, we have developed a new series of imidazo[1,2-b]pyridazines, targeting the calcium-dependent protein kinase-1 (CDPK1) from Toxoplasma gondii. Twenty derivatives were thus synthesized. Structure-activity relationships and docking studies confirmed the binding mode of these inhibitors within the ATP binding pocket of TgCDPK1. Two lead compounds (16a and 16f) were then identified, which were able to block TgCDPK1 enzymatic activity at low nanomolar concentrations, with a good selectivity profile against a panel of mammalian kinases. The potential of these inhibitors was confirmed in vitro on T. gondii growth, with EC50 values of 100 nM and 70 nM, respectively. These best candidates also displayed low toxicity to mammalian cells and were selected for further in vivo investigations on murine model of acute toxoplasmosis.
Subject(s)
Antiprotozoal Agents/pharmacology , Calcium/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Pyridazines/pharmacology , Toxoplasma/drug effects , Toxoplasma/enzymology , Animals , Antiprotozoal Agents/chemical synthesis , Antiprotozoal Agents/chemistry , Dose-Response Relationship, Drug , Drug Design , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyridazines/chemical synthesis , Pyridazines/chemistry , Structure-Activity Relationship , Swine , Toxoplasma/growth & developmentABSTRACT
Neuroblastoma (NB), the most frequent extracranial solid tumor of children accounting for nearly 15% of all childhood cancer mortality, displays overexpression of antiapoptotic Bcl-2 and Mcl-1 in aggressive forms of the disease. The clinical phase 2 drug roscovitine (CYC202, seliciclib), a relatively selective inhibitor of cyclin-dependent kinases (CDKs), and CR8, a recently developed and more potent analog, induce concentration-dependent apoptotic cell death of NB cells (average IC(50) values: 24.2 µM and 0.4 µM for roscovitine and CR8, respectively). Both roscovitine and CR8 trigger rapid down-regulation of the short-lived survival factor Mcl-1 in the 9 investigated human NB cell lines. This effect was further analyzed in the human SH-SY5Y NB cell line. Down-regulation of Mcl-1 appears to depend on inhibition of CDKs rather than on interaction of roscovitine and CR8 with their secondary targets. CR8 is an adenosine triphosphate-competitive inhibitor of CDK9, and the structure of a CDK9/cyclin T/CR8 complex is described. Mcl-1 down-regulation occurs both at the mRNA and protein levels. This effect can be accounted for by a reduction in Mcl-1 protein synthesis, under stable Mcl-1 degradation conditions. Mcl-1 down-regulation is accompanied by a transient increase in free Noxa, a proapoptotic factor. Mcl-1 down-regulation occurs independently of the presence or up-regulation of p53 and of the MYCN status. Taken together, these results suggest that the clinical drug roscovitine and its novel analog CR8 induce apoptotic tumor cell death by down-regulating Mcl-1, a key survival factor expressed in all NB cell lines. CDK inhibition may thus constitute a new approach to treat refractory high-risk NB.