Human Sulfotransferase Assays With PAPS Production in situ.

Yanan Sun,Maria Kristina Parr,Lukas Corbinian Harps,Matthias Bureik

doi:10.3389/fmolb.2022.827638

Yanan Sun, Maria Kristina Parr + Show 2 more

Open Access

https://doi.org/10.3389/fmolb.2022.827638

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Abstract

For in vitro investigations on human sulfotransferase (SULT) catalyzed phase II metabolism, the costly cofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) is generally needed. In the present study, we developed and optimized a new approach that combines SULT-dependent biotransformation using recombinant and permeabilized fission yeast cells (enzyme bags) with PAPS production in situ applying quality by design principles. In the initial application of the procedure, yeast cells expressing human SULT1A3 were used for the production of 4′-hydroxypropranolol-4-O-sulfate from 4-hydroxypropranolol. The optimized protocol was then successfully transferred to other sulfonation reactions catalyzed by SULT2A1, SULT1E1, or SULT1B1. The concomitant degradation of some sulfoconjugates was investigated, and further optimization of the reaction conditions was performed in order to reduce product loss. Also, the production of stable isotope labelled sulfoconjugates was demonstrated utilizing isotopically labelled substrates or 34S-sulfate. Overall, this new approach results in higher space-time yields while at the same time reducing experimental cost.

Highlights

The study of metabolic pathways of drug substances in humans relies both on in vivo and in vitro experiments
A standard protocol was established which allows substrate sulfonation with human SULTs recombinantly expressed in fission yeast using ATP and (NH4)2SO4 instead of PAPS
While production and regeneration of PAPS were previously described in a chemoenzymatic approach (An et al, 2017), in an enzymatic approach (Burkart et al, 2000), and in liver S9 fraction-based biosynthesis (Weththasinghe et al, 2018), this assay successfully combines both, PAPSgeneration and sulfonation of xenobiotics

Summary

Introduction

The study of metabolic pathways of drug substances in humans relies both on in vivo and in vitro experiments. The main specimen for excretion of most drugs and metabolites is urine. In toxicological, forensic or doping control analysis metabolites are often used as target analytes in urine samples (Balcells et al, 2017; Esquivel et al, 2019). In the last two decades, modern in vitro techniques became a viable alternative and a great extension to in vivo studies (Ekins et al, 2000). The majority of phase II metabolites are glucuronide- or sulfoconjugates. The formation of the latter species is catalyzed

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