3 research outputs found
Expression and purification of active human receptor interacting protein 1 kinase using a baculovirus system
Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-?) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1–3 mg of highly purified RIP1 kinase was typically obtained from a 1 L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors
Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system
Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight
MELK
kinase has been implicated in playing an important role in
tumorigenesis. Our previous studies suggested that MELK is involved
in the regulation of cell cycle and its genetic depletion leads to
growth inhibition in a subset of high MELK-expressing basal-like breast
cancer cell lines. Herein we describe the discovery and optimization
of novel MELK inhibitors <b>8a</b> and <b>8b</b> that
recapitulate the cellular effects observed by short hairpin ribonucleic
acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered
a novel fluorine-induced hydrophobic collapse that locked the ligand
in its bioactive conformation and led to a 20-fold gain in potency.
These novel pharmacological inhibitors achieved high exposure in vivo
and were well tolerated, which may allow further in vivo evaluation