a) Simplified inositol pyrophosphate metabolism with focus on kinases. PPIP5K: Inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinase. *The PP-InsP dephosphorylation is catalyzed by nudix hydrolases DIPP1/2α/2β/3.[18] b) Chemical structures and properties of selected IP6K inhibitors: widely used TNP, isozyme-selective Barrow-24, and most potent inhibitor SC-919.

a) Sequence alignment of InsP kinases to identify the gatekeeper position of human IP6Ks. The gatekeeper residue is highlighted in orange. b) Kinase reaction of IP6Ks using uniformly 13C-labeled InsP6 as substrate. Asterisks indicate 13C-labeled positions. The conversion to 5PP-InsP5 was followed by an established spin-echo difference NMR method.[27b, 29] c) Catalytic activity of IP6K gatekeeper mutants indicated by apparent turnover numbers kcat, app. d) Michaelis-Menten graphs for IP6K1wt and IP6K1L210V. e) Screening of established analog-sensitive kinase inhibitors (Figure S4) at 10 μM concentration against IP6K1wt and IP6K1L210V using the NMR assay. All data points were measured in independent triplicates and error bars represent the standard deviation.

a) ATP synthase reaction used for the high-throughput screen. The generation of ATP was monitored by the luminescence-based Kinase-Glo® assay. b) Reduction of putative hits by application of specific thresholds and manual selection. c) Scatter plot comparing the potency of compounds measured in the primary screen against IP6K1wt and IP6K1L210V. Hits highlighted in magenta represent known IP6K inhibitors, or promising hits. 1: 6-hydroxy-dl-DOPA, 2: FMP-201300, 3: myricetin, 4: quercetin. d) Examples of true positive hits that display selectivity for IP6K1L210V, including known IP6K inhibitor quercetin and newly discovered FMP-201300.

a) IC50 curves of FMP-201300 against IP6K1wt and IP6K1L210V. b) IC50 curves of FMP-201300 against IP6K2wt and IP6K2L210V. 100% activity corresponds to the DMSO control and 0% indicates no substrate conversion. c) Inhibitory activity of FMP-201300 analogs against IP6K1L210V at 10 μM concentration. d) Lineweaver-Burk plot of FMP-201300 against IP6K1L210V at two different inhibitor concentrations. The plotted lines for the DMSO control and two different inhibitor concentrations intersect almost precisely on the x-axis, indicating no change in KM value and a decrease in vmax upon inhibition e) IC50 curves of FMP-201300 against IP6K1L210V at two different InsP6 concentrations. All data points were measured in independent triplicates and error bars represent the standard deviation.

HDX-MS reveals increased flexibility in the β2-β3 strands and αC helix induced by the IP6K1L210V mutation that sensitize the enzyme to FMP-201300. A: HDX differences in IP6K1. Time course of deuterium incorporation for a selection of peptides. Raw data can be found in the supplemental file. B: Overall HDX-MS changes in deuterium incorporation induced by the IP6K1L210V mutation. Differences in deuterium exchange rates mapped on a model of IP6K1wt (Alpha-Fold structure prediction Q92551 with ATP and InsP6 from EhIP6KA docked in the active site (PDB: 4O4F)). Peptides that showed significant differences in HDX and met the cut-offs were included (>6% deuterium incorporation and 0.5 Da with an unpaired student t-test of p<0.05). Strongly disordered regions and regions of low per-residue confidence scores (pLDDT) were omitted for clarity. C: A magnified view of gatekeeper region included the β2-β3 strands and αC helix. The same regions that show increased flexibility in the IP6K1L210V mutant (top panel) also undergo large decreases in exchange upon binding to FMP-201300 (bottom panel). This increased flexibility likely allows accommodation of the inhibitor and ATP.