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<p>Sulfonation is a widespread biological reaction catalyzed by a supergene family of enzymes called sulfotransferases (SULTs). SULTs utilize 3’-phosphoadenosine-5’-phospho-sulfate (PAPS) as the universal sulfonate donor to conjugate with a diverse range of endo- and xenobiotic substrates, including neurotransmitters, hormones, and drugs resulting in altering their biological activity. This reaction serves as a major detoxification pathway as conjugation with a sulfonate group renders substrates more hydrophilic and facilitates excretion. Therefore, this process is responsible for reducing the bioavailability of some drugs. In some cases, sulfo-conjugation causes the bio-activation of pro-mutagens and pro-carcinogens, leading to SULTs being risk factors in some cancers. Despite the biological relevance, understanding of this family of enzymes is still scarce. One SULT member that is the focus of the studies described herein is human sulfotransferase 2B1b (SULT2B1b), which had been identified as a potential drug target in prostate cancer. However, the inconsistency in reported kinetic data obtained using radiolabeled assays and the lack of robust assays have become significant limitations for SULT2B1b-targeted drug discovery studies. A label-free assay was developed to bridge this knowledge gap that directly quantifies SULT2B1b sulfonated products. This novel assay utilized high-throughput technology based on Desorption Electrospray Ionization Mass Spectrometry (DESI-MS). Results obtained from the DESI-MS-based assay were compared with those from a fluorometric, coupled-enzyme assay already developed in the Mesecar lab. Both methods provided consistent kinetic data for the reaction of SULT2B1b. Therefore, this novel assay is promising for the application of drug discovery efforts aiming at identifying SULT2B1b inhibitors. The other SULT member studied and described herein is human sulfotransferase 1A1 (SULT1A1), one of humans' most vital detoxifying and drug-metabolizing SULT isoforms that can also be a potential drug target in some cancers. The detailed kinetic mechanism of SULT1A1 was elucidated using steady-state kinetic, product inhibition, dead-end inhibition, and X-crystallographic studies. to gain insights into the role of this enzyme in detoxification, drug metabolism, and the development of inhibitors.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/23745159 |
| Date | 26 July 2023 |
| Creators | Yamasingha Pathiranage Kulathunga (16384296) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/KINETIC_AND_MECHANISTIC_CHARACTERIZATION_OF_HUMAN_SULFOTRANSFERASES_SULT2B1b_AND_SULT1A1_DRUG_TARGETS_TO_TREAT_CANCERS/23745159 |
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