Inositol Thiophosphates as Inhibitors of Mammalian, Plant, and Fungal Phytases

Inositol phosphates constitute a conserved family of signaling molecules that regulate a wide range of cellular processes such as energy metabolism, calcium release, and signal transduction. In mammalian cells, the endoplasmic reticulum-localized enzyme, multiple inositol polyphosphate phosphatase 1 (MINPP1), is the sole phytase known to hydrolyze myo-inositol hexakisphosphate (IP6). MINPP1 is critical for maintaining intracellular inositol phosphate homeostasis, and its dysfunction is linked to severe neurodevelopmental disorders. Beyond mammals, phytases are widely distributed across bacteria, plants, and fungi, where they play essential roles in phosphorus recycling and have important commercial applications in agriculture and animal feed by improving nutrient availability. Despite this broad biological and economic significance, selective chemical tools to study MINPP1 and other phytases, as well as to probe their activity, remain limited. Here, we report the evaluation of the inositol thiophosphates, myo-inositol pentakisthiophosphate (IT5) and hexakisthiophosphate (IT6), as inhibitors of phytases. We show that IT5 and IT6 are resistant to MINPP1 hydrolysis, whereas the native substrates are readily processed, with myo-inositol pentakisphosphate (IP5) hydrolyzed faster than IP6. Both IT5 and IT6 act as potent MINPP1 inhibitors, with nanomolar IC₅₀ values, and display enhanced binding affinities and slower dissociations relative to IP6. Additionally, IT6 inhibits phytases from the plant and fungal kingdoms, including wheat, yeast, and Aspergillus niger, demonstrating cross-kingdom inhibitory activity. Together, these results establish IT6 as a high-affinity, nonhydrolyzable phytase inhibitor and position IT6 as a useful chemical probe for the study of phytases.