Screening method toward ClbP-specific inhibitors

• Published in: Genes and Environment 2023, Vol.45 (8), p.1-7
• Main Authors: Tao Zhou, Takayuki Ando, Akihiro Kudo, Michio Sato, Noriyuki Miyoshi, Michihiro Mutoh, Hideki Ishikawa, Keiji Wakabayashi, Kenji Watanabe
• Format: Article
• Language: Japanese
• ISSN: 1880-7046

[Abstract][Background] Colibactin is a genotoxin produced by Escherichia coli and other Enterobacteriaceae that is believed to increase the risk of colorectal cancer (CRC) of their symbiosis hosts, including human. A peptidase ClbP is the key enzyme for activation of colibactin. Inhibition of ClbP is considered to impede maturation of precolibactin into genotoxic colibactin.Therefore, ClbP-specific inhibitors could potentially prevent the onset of CRC, one of the leading causes of cancer-related deaths in the world.This study intends to establish an efficient screening system for identifying inhibitors that are specific to ClbP. [Methods] Two types of assays were applied in the screening procedure: a probe assay and an LC-MS assay. For the probe assay, we employed the synthesized probe which we described in our previous report. This probe can be hydrolyzed efficiently by ClbP to release a fluorophore. Hence it was applied here for detection of inhibition of ClbP. For the LC-MS assay, formation of the byproduct of precolibactin maturation process, N-myristoyl-D-asparagine, was quantified using a liquid chromatography-mass spectrometry (LC-MS) technique.The probe assay can be performed much faster, while the LC-MS assay is more accurate. Therefore, our method employed the two assays in sequence to screen a large number of compounds for inhibition of ClbP. [Results] A library of 67,965 standard compounds was evaluated by the screening method established in the current study, and one compound was found to show a moderate inhibitory activity against ClbP. [Conclusion] A simple screening method for ClbP-specific inhibitors was established. It was proven to be reliable and is believed to be useful in developing potential prophylactic agents for CRC.