Abstract 5155: RapidFire MS/MS enables both rapid evaluation of multiple histone methyltransferases and label-free high throughput screening of targeted compound libraries
Authors: Bingham Patrick J. , Maegley Karen , Krivacic Cody T. 
Published: May 2015

Introduction: Histone methyltransferases (HMT's) are histone-modifying enzymes, that catalyze the transfer of one, two, or three methyl groups from S-adenosyl-L-methionine (SAM) to lysine and arginine residues on histone proteins. This methylation results in changes in gene expression. Over-expression and misregulation of these enzymes has been associated with cancer, making HMT's an attractive area for the development of new cancer therapeutics. We developed highly sensitive, high throughput RapidFire-based MS/MS biochemical assays to quantitate the enzymatic activity of three different HMT's. This methodology allows a label-free, non-radioactive and coupling enzyme free system to biochemically characterize the kinetic mechanism of HMT's and determine the mechanism of inhibition of small molecule inhibitors.

Methods: The API4000 mass spectrometer coupled with an Agilent RapidFire 300 Mass Spectrometry system was used to rapidly evaluate the enzymatic activity of HMT's using native substrates for both enzymatic characterization of the target as well as screening highly specialized and targeted compound libraries. The Agilent type D column was used for separation of detected products, S-adenosyl-L-homocysteine (SAH) and SAM from other assay components. Electrospray ionization multiple reaction monitoring positive polarity scan was used to detect the two desired analytes of the reaction. SAH was detected using a Q1 mass set to 399.2 amu and a Q3 mass set to 250.1 amu while SAM was detected using a Q1 mass set to 385.2 amu and a Q3 mass set to 136.1 amu.

Preliminary data: Pfizer proprietary compound libraries were assembled and screened in a 384-well plate format against multiple HMT's using the RapidFire 300 coupled with an API4000 mass spectrometer. The enzymatic screens using RapidFire LC/MS enable both one platform and one method for simultaneous investigation of multiple different epigenetic targets in the portfolio. Total cycle time required to process a single assay well for a single HMT in a 384 well plate is 10 seconds. Therefore, one 384-well plate can be sampled in just under 65 minutes, enabling the processing of more than 7,500 compounds per day. Good correlation and a sizeable reduction in cost/well is evident with the RapidFire MS/MS generated results compared with more traditional assay methods for compound screening such as 3H-SAM scintillation proximity assay and 3H-SAM filter plate assay. This presentation will discuss the results generated from the screening efforts of multiple histone methyltransferases against our methyltransferase targeted libraries and our fragment library, as well as the hit follow-up strategy and mechanistic characterization implemented to ensure high quality lead matter was selected for further investment and follow-up.

Citation Format: Patrick J. Bingham, Karen Maegley, Cody T. Krivacic. RapidFire MS/MS enables both rapid evaluation of multiple histone methyltransferases and label-free high throughput screening of targeted compound libraries. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5155. doi:10.1158/1538-7445.AM2014-5155