Probing Sulfotransferase Binding and Inhibition with Synthetic PAPS Analogs Reveals the Role of the 3′-Phosphate and Informs Molecular Tool Design

Abstract

Sulfotransferases (STs) are a class of enzymes that catalyze the transfer of the SULFURYL GROUP (-SO₃−), most commonly from the universal cofactor 3′-phosphoadenosine 5′-phosphosulfate (PAPS), to nucleophilic acceptors bearing hydroxyl or amino groups. Although this process is widespread in biology, it involves the energy-intensive synthesis of PAPS and the generation of a natural inhibitor of sulfotransferases – the bis-phosphorylated nucleotide 3′-phosphoadenosine 5′-phosphate (PAP). This study aims to provide a deeper insight into the structure-activity relationship of the PAPS-ST interaction and lay the groundwork for PAPS-derived molecular tool design. To this end, we report a library of chemically modified PAPS and PAP analogs and evaluate their binding and inhibitory properties toward the plant sulfotransferase AtSOT18 and mammalian SULT1A3. Using microscale thermophoresis, it was found that STs do not differentiate between the 5’-phosphosulfate and 5’-diphosphate analogs, making the latter non-functional mimics potentially useful for structural studies and revealing the critical role of the 3'-phosphate in ensuring the binding specificity. The modifications at the 2’-, 5’- sites, or at the N6 and C8 positions of the adenine rarely made significant contributions to the stabilization of the complex. A strong preference for the adenine base over alternative nitrogenous bases in the cofactor structure was, however, observed. The use of ¹⁹F NMR spectroscopy as a molecular tool for screening potential inhibitors of AtSOT18 and SULT1A3 highlighted C8-(1-amino-2-azidoethyl)-substituted analogs as promising compounds for targeting STs. Overall, the study revealed several novel aspects of molecular recognition of PAPS by STs and layed groundwork for future molecular tool design.