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An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor.

ABSTRACT: Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the αC helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases.

SUBMITTER: Aguirre T

PROVIDER: S-EPMC10578927 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor.

Aguirre Tim T Dornan Gillian L GL Hostachy Sarah S Neuenschwander Martin M Seyffarth Carola C Haucke Volker V Schütz Anja A von Kries Jens Peter JP Fiedler Dorothea D

eLife 20231016

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300 ...[more]

PMID: 37843983

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Project description:Inositol pyrophosphates have been implicated in cellular signaling and membrane trafficking, including synaptic vesicle (SV) recycling. Inositol hexakisphosphate kinases (IP6Ks) and their product, diphosphoinositol pentakisphosphate (PP-IP5 or IP7), directly and indirectly regulate proteins important in vesicle recycling by the activity-dependent bulk endocytosis pathway (ADBE). In the present study, we show that two isoforms, IP6K1 and IP6K3, are expressed in axons. The role of the kinases in SV recycling are investigated using pharmacologic inhibition, shRNA knockdown, and IP6K1 and IP6K3 knockout mice. Live-cell imaging experiments use optical reporters of SV recycling based on vesicular glutamate transporter isoforms, VGLUT1- and VGLUT2-pHluorins (pH), which recycle differently. VGLUT1-pH recycles by classical AP-2 dependent endocytosis under moderate stimulation conditions, while VGLUT2-pH recycles using AP-1 and AP-3 adaptor proteins as well. Using a short stimulus to release the readily releasable pool (RRP), we show that IP6K1 KO increases exocytosis of both VGLUT1-and VGLUT2-pH, while IP6K3 KO decreases the amount of both transporters in the RRP. In electrophysiological experiments we measure glutamate signaling with short stimuli and under the intense stimulation conditions that trigger bulk endocytosis. IP6K1 KO increases synaptic facilitation and IP6K3 KO decreases facilitation compared to wild type in CA1 hippocampal Schaffer collateral synapses. After intense stimulation, the rate of endocytosis of VGLUT2-pH, but not VGLUT1-pH, is increased by knockout, knockdown, and pharmacologic inhibition of IP6Ks. Thus IP6Ks differentially affect the endocytosis of two SV protein cargos that use different endocytic pathways. However, while IP6K1 KO and IP6K3 KO exert similar effects on endocytosis after stimulation, the isoforms exert different effects on exocytosis earlier in the stimulus and on the early phase of glutamate release. Taken together, the data indicate a role for IP6Ks both in exocytosis early in the stimulation period and in endocytosis, particularly under conditions that may utilize AP-1/3 adaptors.

Dataset Information

An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor.

Publications

An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor.

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