Biochimica et biophysica acta · 2016
Leishmania donovani Aurora kinase: A promising therapeutic target against visceral leishmaniasis.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод пока не подготовлен
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Chhajer Rudra, Bhattacharyya Anirban, Didwania Nicky, Shadab Md, Das Nirupam, Palit Partha, Vaidya Tushar, Ali Nahid
Аннотация
BACKGROUND: Aurora kinases are key mitotic kinases executing multiple aspects of eukaryotic cell-division. The apicomplexan homologs being essential for survival, suggest that the Leishmania homolog, annotated LdAIRK, may be equally important.
METHODS: Bioinformatics, stage-specific immunofluorescence microscopy, immunoblotting, RT-PCR, molecular docking, in-vitro kinase assay, anti-leishmanial activity assays, flow cytometry, fluorescence microscopy.
RESULTS: Ldairk expression is seen to vary as the cell-cycle progresses from G1 through S and finally G2M and cytokinesis. Kinetic studies demonstrate their enzymatic activity exhibiting a Km and Vmax of 6.12μM and 82.9pmoles·min(-1)mg(-1) respectively against ATP using recombinant Leishmania donovani H3, its physiological substrate. Due to the failure of LdAIRK-/+ knock-out parasites to survive, we adopted a chemical knock-down approach. Based on the conservation of key active site residues, three mammalian Aurora kinase inhibitors were investigated to evaluate their potential as inhibitors of LdAIRK activity. Interestingly, the cell-cycle progressed unhindered, despite treatment with GSK-1070916 or Barasertib, inhibitors with greater potencies for the ATP-binding pocket compared to Hesperadin, which at nanomolar concentrations, severely compromised viability at IC50s 105.9 and 36.4nM for promastigotes and amastigotes, respectively. Cell-cycle and morphological studies implicated their role in both mitosis and cytokinesis.
CONCLUSION: We identified an Aurora kinase homolog in L. donovani implicated in cell-cycle progression, whose inhibition led to aberrant changes in cell-cycle progression and reduced viability.
GENERAL SIGNIFICANCE: Human homologs being actively pursued drug targets and the observations with LdAIRK in both promastigotes and amastigotes suggest their potential as therapeutic-targets. Importantly, our results encourage the exploration of other proteins identified herein as potential novel drug targets.
METHODS: Bioinformatics, stage-specific immunofluorescence microscopy, immunoblotting, RT-PCR, molecular docking, in-vitro kinase assay, anti-leishmanial activity assays, flow cytometry, fluorescence microscopy.
RESULTS: Ldairk expression is seen to vary as the cell-cycle progresses from G1 through S and finally G2M and cytokinesis. Kinetic studies demonstrate their enzymatic activity exhibiting a Km and Vmax of 6.12μM and 82.9pmoles·min(-1)mg(-1) respectively against ATP using recombinant Leishmania donovani H3, its physiological substrate. Due to the failure of LdAIRK-/+ knock-out parasites to survive, we adopted a chemical knock-down approach. Based on the conservation of key active site residues, three mammalian Aurora kinase inhibitors were investigated to evaluate their potential as inhibitors of LdAIRK activity. Interestingly, the cell-cycle progressed unhindered, despite treatment with GSK-1070916 or Barasertib, inhibitors with greater potencies for the ATP-binding pocket compared to Hesperadin, which at nanomolar concentrations, severely compromised viability at IC50s 105.9 and 36.4nM for promastigotes and amastigotes, respectively. Cell-cycle and morphological studies implicated their role in both mitosis and cytokinesis.
CONCLUSION: We identified an Aurora kinase homolog in L. donovani implicated in cell-cycle progression, whose inhibition led to aberrant changes in cell-cycle progression and reduced viability.
GENERAL SIGNIFICANCE: Human homologs being actively pursued drug targets and the observations with LdAIRK in both promastigotes and amastigotes suggest their potential as therapeutic-targets. Importantly, our results encourage the exploration of other proteins identified herein as potential novel drug targets.
Предметные рубрики MeSH
Adenosine TriphosphateAmino Acid SequenceAnimalsAurora KinasesAza CompoundsCatalytic DomainCell CycleCell SurvivalCytokinesisFemaleIndolesKineticsLeishmania donovaniLeishmaniasis, VisceralMiceMice, Inbred BALB CMolecular Docking SimulationOrganophosphatesProtein Kinase InhibitorsQuinazolinesSulfonamides