Category: Assay Development and Screening

1244-E - A Comprehensive Ultra High-Throughput Screening (UHTS) Campaign to Identify and Characterize Deubiquitinating Enzyme Inhibitors

Wednesday, February 7, 2018
11:30 AM - 12:30 PM

Aaron A. Nazarian1*, Soo Hang Wong1, Carlo J.van Staden1, Jessie St. Martin2,  Steve Schneider2,  Paul Andrews2, David Powers1  and Franck Madoux1

1Amgen Therapeutic Discovery / Discovery Technologies

2Amgen Neuroscience

*Corresponding author:  Aaron A. Nazarian,
Amgen Inc., Thousand Oaks, CA

Ubiquitin plays a central role in mediating protein degradation. Screening for small molecule inhibitors of deubiquitinating (DUB) enzymes may have therapeutic potential for neurodegenerative disease. Here we describe a comprehensive small molecule screening campaign, utilizing our ultra high-throughput screening (UHTS) robotic platform to identify high quality, selective DUB inhibitors. We have developed, miniaturized and used a combination of four different assays to better ascertain the desired inhibition profile. Specifically, we identified primary hits with potential for interference through counter screen, by confirming bona fide activity independent of the detection technology via a competitive binding displacement TRF orthogonal assay, and by ensuring selectivity using a closely related DUB catalytic assay.

Assay miniaturization is an essential component of reducing the cost and duration of small molecule discovery research. The fluorescence polarization (FP) loss of signal assay developed in 384-well format and transferred to UHTS utilizes a TAMRA labeled diubiquitin peptide substrate to monitor DUB enzyme catalytic activity. This assay was limited by a narrow ~50 milliP polarization window. We successfully miniaturized and optimized this DUB FP assay to 1536-well plate format with well-to-well coefficient of variations (CV) values below 2% and plate Z’ greater than 0.75. We realized this UHTS-amenable, high-quality FP assay performance with the additional benefit of a 3-fold decrease in reaction volume from 384-well format to 1536-well format. Assay miniaturization to the 1536-well format reduced reagent consumption for the UHTS primary screen, driving down cost to below $0.01 per well.

This DUB FP assay UHTS primary screen was performed with a throughput of up to 100 assay plates per day. We identified ~300 compounds with IC50s < 10 μM in the DUB FP primary assay, which also exhibited no activity in the FP counter screen assay. As part of our screening strategy, we employed orthogonal and selectivity assays to refine our hit selection and prioritization, ultimately providing validated chemical matter for therapeutic discovery. Thus, here we describe the effective execution of an UHTS campaign using a collection of carefully selected 1536-well assays to interrogate the catalytic and binding activity of a DUB enzyme, with the potential to impact neurodegenerative disease.

Aaron Nazarian

Sr. Associate Scientist
Thousand Oaks, CA

Aaron A. Nazarian is currently Senior Associate Scientist in Therapeutic Discovery at Amgen, where he has made important contributions to the discovery and characterization of various Amgen therapeutic candidates. Aaron has broad experience with assay development and miniaturization, high-throughput screening, and in vitro characterization for both large and small molecules. His strong analytical and automation skills have led to the successful development of binding and functional assays in the fields of G-protein-coupled receptors (GPCRs), ion channels and kinases.

Currently a core member of the Ultra High-Throughput Screening (UHTS) group within Discovery Technologies, Aaron directly contributes to multiple high-quality small molecule screening campaigns and was involved with the implementation of Amgen’s 1536-well format capable UHTS platform. In the Bioassay & Profiling group, Aaron enhanced Amgen’s biologics identification and characterization capabilities. Specifically, he developed a high-capacity T-cell dependent cellular cytotoxicity (TDCC) assay platform, screening over 15 Oncology targets and accelerating Amgen’s discovery pipeline of Bispecific T-Cell Engaging (BiTE) antibodies.

Prior to joining Amgen in 2005, Aaron conducted research studies to elucidate the molecular mechanisms of mammalian gene regulation in the immune system in the lab of Stephen T. Smale (UCLA/ HHMI). Aaron received his Bachelor of Science (BS) degree, magna cum laude, in Microbiology, Immunology & Molecular Genetics (MIMG) from The University of California, Los Angeles (UCLA) in 2003.