Isoform-specific therapeutic control of sulfonation in humans

Abstract

The activities of hundreds, perhaps thousands, of metabolites are regulated by human cytosolic sulfotransferases (SULTs) – a 13-member family of disease relevant enzymes that catalyze transfer of the sulfuryl moiety (-SO3) from PAPS (3′-phosphoadenosine 5′-phosphosulfonate) to the hydroxyls and amines of acceptors. SULTs harbor two independent allosteric sites, one of which, the focus of this work, binds non-steroidal anti-inflammatory drugs (NSAIDs). The structure of the first NSAID-binding site – that of SULT1A1 – was elucidated recently and homology modeling suggest that variants of the site are present in all SULT isoforms. The objective of the current study was to assess whether the NSAID-binding site can be used to regulate sulfuryl transfer in humans in an isoform specific manner. Mefenamic acid (Mef) is a potent (Ki 27 nM) NSAID-inhibitor of SULT1A1 – the predominant SULT isoform in small intestine and liver. Acetaminophen (APAP), a SULT1A1 specific substrate, is extensively sulfonated in humans. Dehydroepiandrosterone (DHEA) is specific for SULT2A1, which we show here is insensitive to Mef inhibition. APAP and DHEA sulfonates are readily quantified in urine and thus the effects of Mef on APAP and DHEA sulfonation could be studied non-invasively. Compounds were given orally in a single therapeutic dose to a healthy, adult male human with a typical APAP-metabolite profile. Mef profoundly decreased APAP sulfonation during first pass metabolism and substantially decreased systemic APAP sulfonation without influencing DHEA sulfonation; thus, it appears the NSAID site can be used to control sulfonation in humans in a SULT-isoform specific manner.

The Human UGT2B7 Nanodisc

“…Hexokinase (yeast) was purchased from Roche Applied Science. Human SULT1E1, SULT2A1, PreScission Protease and GST4A were synthesized as described previously (Zhang et al, 1998;Cook et al, 2019). TnT T7 Insect Cell Extract Protein Expression System was purchased from Promega.…”

“…The MSP1D1 coding region in p28a was purchased from Addgene. Using Gibson assembly (Gibson et al, 2009), the coding region was transferred from p28a to pGEX-6P (Cook et al, 2017(Cook et al, , 2019, which attaches a PreScission-Protease cleavable, MBP/GST/HIS-tag to the MSP1D1 amino terminus.…”

Small-molecule control of neurotransmitter sulfonation

“…Initial Rate. Initial-rate inhibition parameters were obtained from classical initial-rate studies (16,28,43). Reactions were initiated by addition of PAPS (0.50 mM, 17 x K m ) to a solution containing SULT1A3 (20 nM, active sites), inhibitor (0.2 -20 × K i ), 1-HP (5.0 µM, 60 x K m ), and KPO 4 (50 mM), pH 7.5, 25 ± 2°C.…”

“…In previous work (15), we discovered CMP8 (Fig 1), which binds tightly (K i = 34 nM) to SULT1A3 and allosterically inhibits its turnover. The structure of the ligand-bound enzyme (16) revealed that the inhibitor binds at a site that is unique to the 1A3 isoform. The site is situated outside the active site near the so-called catalytic cap of the enzyme (17)(18)(19), which must open and close during the catalytic cycle (20).…”

An Activity-Based Fluorescent Sensor for the Detection of the Phenol Sulfotransferase SULT1A1 in Living Cells

“…[24][25][26][27][28] If radiolabeling is not preferred or if the sulfurylated product does not have an optical signature, mass spectrometry or nuclear magnetic resonance spectroscopy can be used. 20,24,25,29,30 A limited number of these approaches have been translated to living cells but do not provide a direct readout with a spatially and temporally-resolved map of activity. 31 We envision that activity-based fluorescent sensors can address this gap.…”