The sulfotransferase (SULT) 2B1b, which catalyzes the sulfonation of 3β-hydroxysteroids, has been identified as a potential target for prostate cancer treatment. However, a major limitation for SULT2B1b-targeted drug discovery is the lack of robust assays compatible with high-throughput screening and inconsistency in reported kinetic data. For this reason, we developed a novel label-free assay based on high-throughput (>1 Hz) desorption electrospray ionization mass spectrometry (DESI-MS) for the direct quantitation of the sulfoconjugated product (CV<10 %; <1 ng analyte). The performance of this DESI-based assay was compared against a new fluorometric coupled-enzyme method that we also developed. Both methodologies provided consistent kinetic data for the reaction of SULT2B1b with its major substrates, indicating the affinity trend pregnenolone>DHEA>cholesterol, for both the phospho-mimetic and wild-type SULT2B1b forms. The novel DESI-MS assay developed here is likely generalizable to other drug discovery efforts and is particularly promising for identification of SULT2B1b inhibitors with potential as prostate cancer therapeutics.
Keywords: fluorescence; high-throughput screening; mass spectrometry; steroids; sulfotransferases.
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