Fluorinated Phosphoadenosine 5'-Phosphosulfate Analogues for Continuous Sulfotransferase Activity Monitoring and Inhibitor Screening by 19F NMR Spectroscopy.

Agnieszka Mlynarska-Cieslak,Jacek Jemielity,Marcelina Bednarczyk,Mikolaj Chrominski,Joanna Kowalska,Tomasz Spiewla,Marek R Baranowski

doi:10.1021/acschembio.1c00978

Abstract

Sulfotransferases (STs) are ubiquitous enzymes that participate in a vast number of biological processes involving sulfuryl group (SO3) transfer. 3′-phosphoadenosine 5′-phosphosulfate (PAPS) is the universal ST cofactor, serving as the “active sulfate” source in cells. Herein, we report the synthesis of three fluorinated PAPS analogues that bear fluorine or trifluoromethyl substituents at the C2 or C8 positions of adenine and their evaluation as substitute cofactors that enable ST activity to be quantified and real-time-monitored by fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy. Using plant AtSOT18 and human SULT1A3 as two model enzymes, we reveal that the fluorinated PAPS analogues show complementary properties with regard to recognition by enzymes and the working 19F NMR pH range and are attractive versatile tools for studying STs. Finally, we developed an 19F NMR assay for screening potential inhibitors against SULT1A3, thereby highlighting the possible use of fluorinated PAPS analogues for the discovery of drugs for ST-related diseases.

Highlights

Sulfotransferases (STs) are enzymes that catalyze the transfer of sulfuryl (SO3) groups to various nucleophilic acceptors.[1]
We aimed to develop a generally applicable 19 nuclear magnetic resonance (19F NMR)-based assay employing fluorinated phosphoadenosine 5′-phosphosulfate (PAPS) analogues suitable for sulfotransferase activity monitoring and screening both at a single time point
Analogue 1 has a single fluorine substituent and quality of the 19F NMR signal; it may affect the at the C2-position of adenine, whereas 2 and 3 each contains a shape and conformation of the PAPS molecule to some extent, C

Summary

Introduction

Sulfotransferases (STs) are enzymes that catalyze the transfer of sulfuryl (SO3) groups to various nucleophilic acceptors.[1] Sulfotransferase-catalyzed reactions occur in all living domains, including bacteria, plants, and animals, and are involved in a variety of processes, such as enzyme regulation, detoxification, regulating hormonal balance, molecular recognition, and cellular signaling. Several human STs have been identified as biomarkers linked to cancer,[2] neurodegenerative diseases,[3,4] immune response effectiveness,[5] and multiple other disorders.[6]. The vast majority of STs, including mammalian sulfotransferases (SULTs) and plant sulfotransferases (SOTs), use 3′phosphoadenosine 5′-phosphosulfate (PAPS) as a universal cofactor, that is, a sulfate group donor. STs transfer the sulfuryl group from PAPS to acceptor molecules bearing O- or N-. Nucleophilic functional groups and release PAP as the byproduct (Figure 1A). ST substrate specificity varies from small molecules to complex macromolecules, including proteins and proteoglycans

Objectives

Methods

Results