Abstract 1757: Developing sensitive assays to facilitate hit identification of RNA modifying enzymes

Abstract

Abstract N-6-methyladenosine (m6A) is the most abundant RNA chemical modification found in the eukaryotic transcriptome. It is a dynamic and reversible modification which plays a role in RNA function across a wide range of biological processes. Dysregulation of the m6A modification and the readers, writers and erasers involved in these processes have been implicated in the progression of multiple types of cancer and metabolic disease. RNA modifying enzymes are therefore emerging as exciting therapeutic targets. To facilitate and accelerate drug discovery efforts in this area, we have developed several sensitive and robust assays suitable for identifying and assessing chemical matter against the RNA modifying enzymes, fat mass and obesity associated protein (FTO) and methyltransferase-like 3/14 (METTL3/14). Specifically, a robust Microscale Thermophoresis (MST) assay was developed, validated, and used to screen 100 compounds from the LOPAC for novel human FTO small molecule binders. Five compounds, which displayed reproducible, concentration-dependent binding in the MST screen, were tested for inhibition of human FTO RNA demethylation activity and revealed a single, novel FTO inhibitor. To further profile emerging FTO hit matter, we are also developing sensitive and robust mass spectrometry assays to demonstrate target inhibition and proof of mechanism. Our progress in this area will be reported. Additionally, we have developed a highly sensitive and robust LC-MS/MS assay to monitor the METTL3/14 catalysed conversion of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). This assay is suitable for identifying either substrate competitive or allosteric METTL3/14 inhibitors. In summary, our proof of concept studies describe the development of several robust and sensitive assays suitable for hit identification and further characterisation of inhibitors of key RNA modifying enzymes and offer a platform for the discovery and development of novel therapeutics in this emerging area of research. Citation Format: Chris Tomlinson, Rachel Lawrence, Stuart Best, Grace Boden, Thomas Alanine, James Wasp, Bethany Smith, Ross Wilkie, Stuart Thomson, John Unitt, Scott Pollack, Allan M. Jordan. Developing sensitive assays to facilitate hit identification of RNA modifying enzymes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1757.