This proposal uses an innovative chemoproteomic technology termed isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) to map the druggable hotspots targeted by withaferin A, a natural product derived from Acnistus arborescens that bears a cysteine-reactive Michael acceptor and impairs TNBC cell viability. IsoTOP-ABPP uses reactivity-based chemical probes to map proteome-wide reactive, functional, and druggable hotspots directly in complex proteomes. When used in a competitive manner, covalently-acting small-molecules can be competed against corresponding reactivity-based probes to enable target discovery. Using isoTOP-ABPP platforms, this proposal shows preliminary data for identifying multiple cysteine hotspots targeted by withaferin A, including C377 of PPP2R1A, the regulatory subunit of the major tumor suppressor protein phosphatase 2A (PP2A). The preliminary data show that withaferin A treatment in TNBC cells impairs phosphorylation of the major PP2A substrate and oncogenic signaling protein AKT, potentially explaining its potent anti- cancer activity in these cells. Using fragment-based covalent ligand discovery methods and isoTOP- ABPP platforms, a cysteine-reactive acrylamide lead DKM 2-90 has also been identified that more selectively targets PP2A, impairs phospho-AKT signaling, and impairs TNBC cell viability. This proposal hypothesizes that withaferin A impairs TNBC pathogenicity through targeting a cysteine hotspot in PPP2R1A to activate PP2A activity and impair oncogenic signaling pathways such as AKT. This proposal will use isoTOP-ABPP platforms to investigate the mechanisms of action of withaferin A in impairing TNBC pathogenicity and use covalent ligand discovery approaches to develop more synthetically accessible potent and selective modulators against withaferin A targets.

Public Health Relevance

In this proposal, I will use chemoproteomics platforms to investigate the mechanisms of action of withaferin A in impairing breast cancer pathogenicity and use covalent ligand discovery approaches to develop more synthetically accessible potent and selective modulators against withaferin A targets.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31CA225173-02
Application #
9679232
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Radaev, Sergey
Project Start
2018-05-01
Project End
2019-11-30
Budget Start
2019-05-01
Budget End
2019-11-30
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Chemistry
Type
Graduate Schools
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94710
Grossman, Elizabeth A; Ward, Carl C; Spradlin, Jessica N et al. (2017) Covalent Ligand Discovery against Druggable Hotspots Targeted by Anti-cancer Natural Products. Cell Chem Biol 24:1368-1376.e4