Biology subjects

Blind, R. D.

Publications and source records attributed to Blind, R. D..

5 recordsLinked to original sources

bioRxiv2025

Structural rationalization of IPMK inhibitor potency.

Inositol polyphosphate multikinase (IPMK) is a kinase linked to several cancers, recent development of a large panel of ATP-competitive inhibitors has reinvigorated enthusiasm for targeting IPMK. However, the structural basis for how these inhibitors achieve high potency is unknown. Here, we report 14 novel co-crystal structures (1.7[A] - 2.0[A] resolution) of human IPMK kinase domain with these inhibitors. We also apply a radiolabeled assay and isothermal titration calorimetry that permit high-confidence IC50 and KD value determinations. The structures reveal a pocket in the ATP-binding site engaged by the most potent inhibitors. Two ordered waters also participate in hydrogen-bonding networks associated with the most potent inhibitors. In addition to providing the molecular basis for observed increases in potency and selectivity, the data presented here provide a toolbelt of 14 novel inhibitor-bound structures of human IPMK that can serve as a reference for all future IPMK structure-based inhibitor development efforts.

biochemistry↗

bioRxiv2024

Design, synthesis and cellular characterization of a new class of IPMK kinase inhibitors

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 InsP5, a direct product of IPMK kinase activity, while having no effect on InsP6 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 InsP5 metabolic signature, and reveals novel aspects inositol phosphate cellular metabolism and signaling.

pharmacology and toxicology↗

bioRxiv2024

X-ray crystallographic analyses of 14 IPMK inhibitor complexes

Inositol polyphosphate multikinase (IPMK) is a ubiquitously expressed kinase that has been linked to several cancers. Here, we report 14 new co-crystal structures (1.7[A] - 2.0[A] resolution) of human IPMK complexed with various IPMK inhibitors developed by another group. The new structures reveal two ordered water molecules that participate in hydrogen-bonding networks, and an unoccupied pocket in the ATP-binding site of human IPMK. New Protein Data Bank (PDB) codes of these IPMK crystal structures are: 8V6W(1.95[A]), 8V6X(1.75[A]), 8V6Y(1.70[A]), 8V6Z(1.85[A]), 8V70(1.85[A]), 8V71(1.70[A]), 8V72(2.0[A]), 8V73(1.90[A]), 8V74(1.85[A]), 8V75(1.85[A]), 8V76(1.95[A]),8V77(1.95[A]), 8V78(1.95[A]), 8V79(1.95[A]).

biochemistry↗

bioRxiv2024

IPMK regulates HDAC3 activity and histone H4 acetylation in human cells.

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.

biochemistry↗

bioRxiv2018

Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity.

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P2 (PIP2). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains ({Delta}IPMK) are presented. Although the KM for PIP2 is identical between {Delta}IPMK and wild type, {Delta}IPMK has a 1.8-fold increase in kcat for PIP2, indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 [A] crystal structure of {Delta}IPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative \"ATP-clamp\" in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the KM for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.

biochemistry↗