Towards the Identification of Human Sulfotransferase 2B1b (SULT2B1) Inhibitors for the Treatment of Prostate Cancer

Qing Zhou, Purdue University


Human sulfotransferase 2B1b (SULT2B1b) is a member in human SULT family. SULT2B1b catalyzes the transfer of a sulfo group from a universal donor 3’- phophpadenosine 5’-phosphosulfate (PAPS) to a 3?-hydroxysteroid acceptor, to pro-duce adenosine 3’,5’-diphosphate (PAP) and a sulfonated hydroxysteroid. 3?-hydroxysteroid substrates that have been identified and used in this study include cholesterol, pregnenolone, and dehydroepiandrosterone (DHEA). The majority of SULT2B1b’s physiological role remains unclear. It has been suggested that SULT2B1b may potentially serve as a drug target for treating prostate cancer. SULT2B1b is expressed in multiple human tissues, including prostate. Moreover, overexpression of SULT2B1b is correlated with production of cholesterol sulfate (a catalytic product of SULT2B1b) in cancerous prostate tissue samples. Using multiple prostate cancer cell lines, it has been found that knocking down SULT2B1b expression through siRNA silencing results in attenuated cell growth. In summary, previous studies indicate that suppressing SULT2B1b is a potential way to attenuate prostate cancer cell growth and a direction to develop prostate cancer therapeutics. Based on previous findings, we propose to identify small-molecule inhibitors of SULT2B1b that would specifically inhibit the catalytic activity of SULT2B1b, decrease the production of cholesterol sulfate, and lead to attenuated prostate cancer cell growth. Meanwhile, the specific inhibitors of SULT2B1b can be quantitatively applied to cells and animal models to facilitate the study of SULT2B1b, such as the pathways that SULT2B1b is involved in. Since very little knowledge is available for specific inhibitors of SULT2B1b, such as preferred scaffolds, we propose to utilize high-throughput screening (HTS) to test a broad diversity of small-molecule compounds. However, the current most commonly used in vitro activity assay for SULT2B1b is a radiometric assay, which is very tedious and cannot be applied in HTS. To start with, we designed an in vitro coupled-enzyme fluorometric activity assay for SULT2B1b, which can conveniently monitor the fluorescence signal due to a fluorescent product continuously. Furthermore, this assay is set up in microplates and can be adapted for HTS. In our study, we first purified and characterized the coupling enzyme SULT1A1. On the basis of understanding the kinetic parameters of SULT1A1, we purified SULT 2B1b and developed a novel activity assay for measuring its activity. Using this activity assay, we performed kinetic characterization for multiple SULT2B1b constructs, including the wild-type SULT2B1b, S348D phosphomimetic mutant, H125A catalytic mutant, and K70A catalytic mutant. Next, we further optimized the activity assay for HTS. Successful attempts include but are not limited to scaling down the reactions to 20 or 50 µL, enzymatically synthesizing one of the substrates PAPS, and adjusting the buffer pH. Using Z’-factor as the criterion to assess the quality of the assay, we achieved an assay in HTS-format with good reliability indicated by Z’-factor higher than 0.7. Facilitated by Bindley Bioscience Center of Purdue University, we performed HTS using the optimized HTS assay to screen totally 27,200 small-molecule compounds for SULT2B1b in two rounds. The assay was robust during screening with Z’-factors higher than 0.5 for all the plates. We identified 47 hits, which were corresponding to 0.17% hit rate. Lastly, we validated the identified HTS hits using various methods, including assessing the inhibition by in vitro coupled-enzyme assay in non-HTS format, evaluating the effects of compounds on prostate cancer cell growth and assessing whether the compounds would alter the ability of prostate cancer cells to produce cholesterol sulfate by desorption electrospray ionization mass spectrometry (DESI-MS). We identified one compound, which is a potent inhibitor of SULT2B1b in vitro with an IC50 value of approximately 20 µM. However, when being tested in prostate cancer cells, this compound slowed down the cell growth by possibly affecting other targets. Using the established workflow, we also studied the role of phosphorylation at serine 348 of SULT2B1b. We conclude that this phosphorylation does not change the catalytic activity of SULT2B1b in vitro and in cellulo. Thus, the significance of this phosphorylation site still needs further investigation. In summary, we established a pipeline for early-phase drug discovery for SULT2B1b, including an in vitro activity assay, an HTS work flow, and tests in prostate cancer cells regarding their growth and capability of producing cholesterol sulfate. The identified one SULT2B1b inhibitor is not ideal for cellular uses, but it can serve as a tool compound to study SULT2B1b in vitro. In the future, we can continue performing HTS to explore other scaffolds, optimize the current hit compound, and develop other assays to study the interactions between identified inhibitors and SULT2B1b.




Mesecar, Purdue University.

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