ABSTRACT
Haspin is a mitotic protein kinase required for proper cell division by modulating Aurora B kinase localisation and activity as well as histone phosphorylation. Here a series of imidazopyridazines based on the CHR-6494 and Structure Activity Relationship was established. An assessment of the inhibitory activity of the lead structures on human Haspin and several other protein kinases is presented. The lead structure was rapidly optimised using a combination of crystal structures and effective docking models, with the best inhibitors exhibiting potent inhibitory activity on Haspin with IC50 between 6 and 100 nM in vitro. The developed inhibitors displayed anti-proliferative properties against various human cancer cell lines in 2D and spheroid cultures and significantly inhibited the migration ability of osteosarcoma U-2 OS cells. Notably, we show that our lead compounds are powerful Haspin inhibitors in human cells, and did not block G2/M cell cycle transition due to improved selectivity against CDK1/CyclinB.
Subject(s)
Antineoplastic Agents/chemical synthesis , Bone Neoplasms/drug therapy , Indazoles/chemical synthesis , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Osteosarcoma/drug therapy , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridazines/chemical synthesis , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , CDC2 Protein Kinase/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin B/metabolism , Drug Screening Assays, Antitumor , Histones/chemistry , Humans , Indazoles/pharmacology , Molecular Docking Simulation , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Pyridazines/pharmacology , Structure-Activity RelationshipABSTRACT
Autophagy is a catabolic process that delivers cytoplasmic components to the lysosomes. Protein modification by ubiquitination is involved in this pathway: it regulates the stability of autophagy regulators such as BECLIN-1 and it also functions as a tag targeting specific substrates to autophagosomes. In order to identify deubiquitinating enzymes (DUBs) involved in autophagy, we have performed a genetic screen in the Drosophila larval fat body. This screen identified Uch-L3, Usp45, Usp12 and Ubpy. In this paper, we show that Ubpy loss of function results in the accumulation of autophagosomes due to a blockade of the autophagy flux. Furthermore, analysis by electron and confocal microscopy of Ubpy-depleted fat body cells revealed altered lysosomal morphology, indicating that Ubpy inactivation affects lysosomal maintenance and/or biogenesis. Lastly, we have shown that shRNA mediated inactivation of UBPY in HeLa cells affects autophagy in a different way: in UBPY-depleted HeLa cells autophagy is deregulated.
Subject(s)
Autophagy , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/enzymology , Endopeptidases/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Lysosomes/metabolism , Organelle Biogenesis , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific Proteases/metabolism , Animals , Biocatalysis , Drosophila melanogaster/ultrastructure , Gene Silencing , Genetic Testing , HeLa Cells , Humans , Mutant Proteins/metabolismABSTRACT
Seven new adociaquinone derivatives, xestoadociaquinones A (1a), B (1b), 14-carboxy-xestoquinol sulfate (2) and xestoadociaminals A-D (3a, 3c, 4a, 4c), together with seven known compounds (5-11) were isolated from an Indonesian marine sponge Xestospongia sp. Their structures were elucidated by extensive 1D and 2D NMR and mass spectrometric data. All the compounds were evaluated for their potential inhibitory activity against eight different protein kinases involved in cell proliferation, cancer, diabetes and neurodegenerative disorders as well as for their antioxidant and antibacterial activities.
