RESUMO
Mechanistic Target of Rapamycin (mTOR) binds the small metabolite inositol hexakisphosphate (IP6) as shown in structures of mTOR, however it remains unclear if IP6, or any other inositol phosphate species, can activate mTOR kinase activity. Here, we show that multiple, exogenously added inositol phosphate species (IP6, IP5, IP4 and IP3) can all enhance the ability of mTOR and mTORC1 to auto-phosphorylate and incorporate radiolabeled phosphate into peptide substrates in in vitro kinase reactions. Although IP6 did not affect the apparent KM of mTORC1 for ATP, monitoring kinase activity over longer reaction times showed increased product formation, suggesting inositol phosphates stabilize an active form of mTORC1 in vitro. The effects of IP6 on mTOR were reversible, suggesting IP6 bound to mTOR can be exchanged dynamically with the free solvent. Interestingly, we also observed that IP6 could alter mTOR solubility and electrophoretic mobility in SDS-PAGE in the presence of manganese, suggesting divalent cations may play a role in inositol phosphate regulation of mTOR. Together, these data suggest for the first time that multiple inositol phosphate species (IP4, IP5 and IP6) can dynamically regulate mTOR and mTORC1 by promoting a stable, active state of the kinase. Our data suggest that studies of the dynamics of inositol phosphate regulation of mTOR are well justified.
RESUMO
Many genetic studies have established the kinase activity of inositol phosphate multikinase (IPMK) is required for the synthesis of higher-order inositol phosphate signaling molecules, the regulation of gene expression and control of the cell cycle. These genetic studies await orthogonal validation by specific IPMK inhibitors, but no such inhibitors have been synthesized. Here, we report complete chemical synthesis, cellular characterization, structure-activity relationships and rodent pharmacokinetics of a novel series of highly potent IPMK inhibitors. The first-generation compound 1 (UNC7437) decreased cellular proliferation and tritiated inositol phosphate levels in metabolically labeled human U251-MG glioblastoma cells. Compound 1 also regulated the transcriptome of these cells, selectively regulating genes that are enriched in cancer, inflammatory and viral infection pathways. Further optimization of compound 1 eventually led to compound 15 (UNC9750), which showed improved potency and pharmacokinetics in rodents. Compound 15 specifically inhibited cellular accumulation of InsP 5 , a direct product of IPMK kinase activity, while having no effect on InsP 6 levels, revealing a novel metabolic signature detected for the first time by rapid chemical attenuation of cellular IPMK activity. These studies designed, optimized and synthesized a new series of IPMK inhibitors, which reduces glioblastoma cell growth, induces a novel InsP 5 metabolic signature, and reveals novel aspects inositol phosphate cellular metabolism and signaling.
RESUMO
Histone deacetylases (HDACs) repress transcription by catalyzing the removal of acetyl groups from histones. Class 1 HDACs are activated by inositol phosphate signaling molecules in vitro , but it is unclear if this regulation occurs in human cells. Inositol Polyphosphate Multikinase (IPMK) is required for production of inositol hexakisphosphate (IP6), pentakisphosphate (IP5) and certain tetrakisphosphate (IP4) species, all known activators of Class 1 HDACs in vitro . Here, we generated IPMK knockout (IKO) human U251 glioblastoma cells, which decreased cellular inositol phosphate levels and increased histone H4-acetylation by mass spectrometry. ChIP-seq showed IKO increased H4-acetylation at IKO-upregulated genes, but H4-acetylation was unchanged at IKO-downregulated genes, suggesting gene-specific responses to IPMK knockout. HDAC deacetylase enzyme activity was decreased in HDAC3 immunoprecipitates from IKO vs . wild-type cells, while deacetylase activity of other Class 1 HDACs had no detectable changes in activity. Wild-type IPMK expression in IKO cells fully rescued HDAC3 deacetylase activity, while kinase-dead IPMK expression had no effect. Further, the deficiency in HDAC3 activity in immunoprecipitates from IKO cells could be fully rescued by addition of synthesized IP4 (Ins(1,4,5,6)P4) to the enzyme assay, while control inositol had no effect. These data suggest that cellular IPMK-dependent inositol phosphates are required for full HDAC3 enzyme activity and proper histone H4-acetylation. Implications for targeting IPMK in HDAC3-dependent diseases are discussed.