Abstract 3149: Desorption electrospray ionization mass spectrometry (DESI-MS) high-throughput screening of small-molecule inhibitors for human sulfotransferase 2B1b &amp; biophysical and crystallographic characterization of target-hit interaction
Authors: Beinan Yang, Nicolás M. Morato, Samadhi Kulathunga, Timothy L. Ratliff, Graham Cooks, Andrew D. Mesecar
Journal: Cancer Research (2025)

Upon malignancy and metastasis to distant organs such as brain, lung and bone marrow, the 5-year survival rate of castration-resistance prostate cancer (CRPC) is only around 30%. Therefore, there is an urgent need for identifying new CRPC drug targets and developing new CRPC treatments. A potential new drug target for CRPC is human sulfotransferase isozyme SULT2B1b. It catalyzes the transfer of the sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to cholesterol to produce cholesterol sulfate (CS). Increased expression of SULT2B1b has been shown to correlate to prostate cancer cell proliferation with decreased level of TNF-mediated apoptosis. Knockdown of SULT2B1b leads to elevation of TNF expression and NF-κB activation. SULT2B1b knockdown also enhances TNF-mediated apoptosis in both TNF-sensitive (LNCaP)and TNF-resistant (C4-2) cells. We demonstrate herein that catalytic knockout mutants, H125A and K70A, of SULT2B1b abolish CS production in cells mimicking genetic knockdown studies. This result suggests that small molecule inhibitors of SULT2B1b activity could serve as potential CRPC therapeutics. Towards this goal, we established a novel high-throughput (50nL, &amp;lt;1s per sample) label-free DESI-MS platform for the screening of small-molecule compounds with excellence Z’ scores ranging from0.73 to 0.86. Using DESI-MS, we first determined Kcat and KM values for catalysis of cholesterol by SULT2B1b to be 0.05 ± 0.002 min-1 and 19.8 ± 2.2 µM respectively. We then screened 4500compounds from three chemical libraries (ChemBridge Kinase Inhibitor, LOPAC 1280 &amp;AnalytiCon MEGx Natural Products) and identified 3 bona fide hit compounds (#099, #170 and#954) that had IC50 values ranging from 1.2 to 1.6 µM. We also showed via DESI-MS that these three compounds, each containing multiple hydroxyl groups, could not be sulfated. NanoDSF was used to show that the hit compounds stabilize the SULT2B1b melting temperature by 1.7°C to3.0°C. Binding interactions of the hit molecules were also examined using microscale thermophoresis (MST) and dissociation constants (KD) of 13.5 µM (#099), 6.9 µM (#170) and 7.2µM (#954) were determined. X-ray structures of SULT2B1b in complex with #099 and #170 were determined to 2.8 Å and 2.9 Å resolution respectively. Both compounds are found bound within the cholesterol substrate binding site. One important structural observation is the N-terminus helix (residues 1 - 28) is displaced due to the binding of the inhibitors. In summary, we used DESI-MS to identify three compounds that potently inhibit SULT2B1b through interactions at the cholesterol binding site. These results should now allow for the structure-based design and development of more potent lead compounds of SULT2B1b which ultimately may be useful forCRPC treatment. Citation Format: Beinan Yang, Nicolás M. Morato, Samadhi Kulathunga, Timothy L. Ratliff, Graham Cooks, Andrew D. Mesecar. Desorption electrospray ionization mass spectrometry (DESI-MS) high-throughput screening of small-molecule inhibitors for human sulfotransferase 2B1b &amp; biophysical and crystallographic characterization of target-hit interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3149.