Biology subjects

Shears, S. B.

Publications and source records attributed to Shears, S. B..

4 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

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↗

bioRxiv2022

Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues

Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly-created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references, and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP5 and 2-PP-InsP5. The latter has not previously been described in Nature. Analysis of feces and the commercial mouse diet suggest the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP5 and 2-PP-InsP5. In summary, our study substantially expands PP-InsP biology in mammals.

biochemistry↗

bioRxiv2020

Analysis of Inositol Phosphate Metabolism by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry (CE-ESI-MS)

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is highly desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables for the first time the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we uncover that there must be unknown inositol synthesis pathways in mammals, highlighting the unique potential of this method to dissect inositol phosphate metabolism and signalling.

biochemistry↗