Thiophosphate Bioisosteres of Inositol Hexakisphosphate Enhances Binding Affinity and Residence Time to Bacterial Virulence Factors.

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