Abstract

The ability to quantitatively measure a small molecule’s interactions with its protein target(s) is crucial for both mechanistic studies of signaling pathways and in drug discovery. However, current methods to achieve this have specific requirements that can limit their application or interpretation. Here we describe a complementary target-engagement method, HIPStA (Heat Shock Protein Inhibition Protein Stability Assay), a high-throughput method to assess small molecule binding to endogenous, unmodified target protein(s) in cells. The methodology relies on the change in protein turnover when chaperones, such as HSP90, are inhibited and the stabilization effect that drug-target binding has on this change. We use HIPStA to measure drug binding to three different classes of drug targets (receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases), via quantitative fluorescence imaging. We further demonstrate its utility by pairing the method with quantitative mass spectrometry to identify previously unknown targets of a receptor tyrosine kinase inhibitor.

Quantitative profiling of small molecule-protein binding in cells can aid basic biochemical research and drug discovery. Here, the authors develop the Heat Shock Protein Inhibition Protein Stability Assay (HIPStA) as a high-throughput method to assess cellular target engagement and identify new drug targets.

Details

Title
Chaperone mediated detection of small molecule target binding in cells
Author
Cho, Kelvin F 1 ; Ma, Taylur P 2 ; Rose, Christopher M 2   VIAFID ORCID Logo  ; Kirkpatrick, Donald S 2   VIAFID ORCID Logo  ; Yu Kebing 2 ; Blake, Robert A 1 

 Genentech Inc., Department of Biochemical and Cellular Pharmacology, South San Francisco, USA (GRID:grid.418158.1) (ISNI:0000 0004 0534 4718) 
 Proteomics & Lipidomics, Genentech Inc., Department of Microchemistry, South San Francisco, USA (GRID:grid.418158.1) (ISNI:0000 0004 0534 4718) 
Publication year
2020
Publication date
2020
Publisher
Nature Publishing Group
e-ISSN
20411723
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2344205887
Copyright
This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.