Thiophosphate bioisosteres of inositol hexakisphosphate enhance binding affinity and residence time on bacterial virulence factors

Inositol phosphates are essential for mammalian cell signalling with critical roles in cellular processes. The fully phosphorylated inositol phosphate, <i>myo</i>-inositol hexakisphosphate (IP6), modulates numerous eukaryotic proteins and bacterial virulence factors. It has been suggested that the high charge density of IP6 causes restructuring of virulence factors in mammalian cells, activating their enzymatic activity. IP6 is challenging to study due to its phytase instability and propensity to precipitate. Here we suggest that the thiophosphate bioisostere, <i>myo</i>-inositol hexakisthiophosphate (IT6), will mitigate these issues, as thiophosphate substitution has been found to be phytase resistant and improve solubility. Assessment of the chemical properties of IT6 has indeed validated these characteristics. In addition, we performed biophysical characterization of IT6 binding to the virulence factors <i>Salmonella enterica</i> serovar Typhimurium AvrA, <i>Vibrio parahaemolyticus</i> VopA, and <i>Clostridioides difficile</i> TcdB. Our data show that the higher charge density of IT6 increased its binding affinity and residence time on the proteins, which improved stabilization of the bound-state. IT6 is a valuable tool for structural biology research and the described biophysical characteristics of thiophosphate substitution are of value in medicinal chemistry.