A Fluorescent, HTS‐Adaptable Coupled‐Enzyme Assay for Measurement of Human Cytosolic Sulfotransferase (SULT) 2B1b Activity and Identification of Small‐Molecule Inhibitors
Abstract
Human cytosolic sulfotransferase (SULT) 2B1b, as a member in the SULT family, catalyzes the transfer of a sulfonate moiety (‐SO3) from the universal donor 3‐phopho‐adenosine 5′‐phophosulfate (PAPS) to the hydroxyl groups of various substrates, which leads to a change in water solubility and thus triggers various downstream effects. SULT2B1b highly prefers 3β‐hydroxysteroids, such as cholesterol, as its substrates. By switching the sulfonation state of its substrates, SULT2B1b is involved in the regulation of steroid synthesis, transportation and signaling. Furthermore, SULT2B1b has been suggested as a potential drug target for prostate cancer. Therefore, we aim to identify small‐molecule inhibitors from high‐throughput screening (HTS), and utilize them to study the physiological roles of SULT2B1b in both normal tissues and carcinogenesis as well as provide possible leads for drug discovery in the treatment of prostate cancer. Considering the lack of convenient and HTS‐adaptable activity assays for SULT2B1b, we have developed a fluorescent, coupled‐enzyme assay with real‐time fluorescence‐monitoring capabilities as opposed to the commonly used endpoint radioactivity assays. The new assay was adapted for HTS in 384‐well plates and after non‐trivial assay optimization, this activity assay resulted in a reliable performance (Z′‐factor=0.8 in preliminary study, Z‐factor=0.6~0.7 in the actual screening). Initial hits from HTS have been further validated in vitro and in cellulo. Our criteria for highly selective inhibitors of SULT2B1b include high potency in vitro, and success in mimicking the effects of SULT2B1b siRNA in cellular studies, including attenuating prostate cancer cell proliferation and decreasing cholesterol sulfate accumulation.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Apr 01, 2016
- Source ID
- 10.1096/fasebj.30.1_supplement.612.14
Entities
People
- Andrew Mesecar
- Qing Zhou
Organizations
- Purdue University
- United States Department of Defense