Abstract 2976: Confirmation of in-cell target engagement using the proteolysis targeting chimeras (PROTACs) against pirin
Authors: Sharp Swee Y. , Chessum Nicola E. , Caldwell John J. , Powers Marissa V. , Pasqua A Elisa , Wilding Birgit , Collins Ian , Ozer Bugra , Rowlands Martin , Stubbs Mark , Burke Rosemary , Montfort Rob L. van , Cheeseman Matthew D. , Clarke Paul A. , Workman Paul , Jones Keith 
Published: August 2018

We recently reported the identification of the original bisamide lead compound CCT251236 as an inhibitor of the HSF1 stress pathway with a high affinity for the putative transcription factor co-regulator, pirin (SPR KD=44nM) (Cheeseman et al., J Med Chem, 60; 180-201, 2017). Pirin is a highly conserved non-heme iron-binding regulatory protein that is a member of the functionally diverse cupin superfamily, but has no known enzymatic function or biomarkers of activity. To understand further this poorly characterized protein and to confirm that CCT251236 binds to pirin within living cells, we conceived and optimized a heterobifunctional protein degradation probe using the proteolysis targeting chimeras (PROTACs; CCT367766) comprising a pirin-binding moiety linked to the cereblon-targeting ligand thalidomide. This PROTAC molecule was designed to recruit pirin to the E3 ubiquitin ligase cereblon resulting in the ubiquitylation and degradation of pirin. Negative control probes lacking binding to pirin (CCT367857) or cereblon (CCT367936) were also designed and synthesized. We demonstrated a concentration-dependent depletion of pirin protein from as low as 0.5nM and as early as 2 hr treatment of SKOV3 human ovarian cancer cells with the PROTAC. The negative controls CCT367857 and CCT367936 exhibited no pirin depletion at equimolar concentrations. At higher concentrations of the active probe, a hook effect is observed, consistent with the formation of a ternary complex. Degradation of pirin by the PROTAC was confirmed to be proteasome-dependent by rescue of depletion following pre-incubation with the proteasome inhibitor MG132. In addition, the PROTAC could not induce pirin degradation in CRISPR/cas9 cereblon knockout SKOV3 cells, confirming dependence on cereblon. Pre-treatment with the bisamide compound CCT251236 or free thalidomide abrogated the PROTAC-induced pirin degradation, consistent with pirin and cereblon engagement. Finally, to estimate the cellular selectivity of the PROTAC to pirin in an unbiased manner, we carried out whole proteome mass spectrometry in SKOV3 cells. From 8547 quantifiable proteins identified, only pirin (2.3-fold reduction) displayed a statistically significant (Padj&lt;0.05) difference in protein expression, indicating impressive selectivity. In summary, we have designed a PROTAC as an intracellular probe against a poorly understood molecular target, pirin. This approach has allowed us to confirm in-cell target engagement of our bisamide lead CCT251236 with pirin and validates CCT367766 as a PROTAC tool to further study this largely unexplored protein. Our results also provide a systematic approach for the use of the powerful PROTAC technology to investigate potential and poorly understood cancer drug targets.

Citation Format: Swee Y. Sharp, Nicola E. Chessum, John J. Caldwell, Marissa V. Powers, A Elisa Pasqua, Birgit Wilding, Ian Collins, Bugra Ozer, Martin Rowlands, Mark Stubbs, Rosemary Burke, Rob L. van Montfort, Matthew D. Cheeseman, Paul A. Clarke, Paul Workman, Keith Jones. Confirmation of in-cell target engagement using the proteolysis targeting chimeras (PROTACs) against pirin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2976.