Translation Termination Factor GSPT1 Is a Phenotypically Relevant

Subscriber access provided by BUPMC - Bibliothèque Universitaire Pierre et Marie Curie

Letter

The translation termination factor GSPT1 is a phenotypically relevant off-target of heterobifunctional phthalimide degraders Mette Ishoey, Someth Chorn, Natesh Singh, Martin G. Jaeger, Matthias Brand, Joshiawa Paulk, Sophie Bauer, Michael A Erb, Katja Parapatics, André C. Müller, Keiryn L. Bennett, Gerhard F. Ecker, James E. Bradner, and Georg E. Winter ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00969 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

The translation termination factor GSPT1 is a phenotypically relevant offtarget of heterobifunctional phthalimide degraders

Mette Ishoey1,# , Someth Chorn2,#, Natesh Singh3, Martin G. Jaeger2, Matthias Brand2, Joshiawa Paulk1,4, Sophie Bauer2, Michael A. Erb1, Katja Parapatics2, André C. Müller2, Keiryn L. Bennett2, Gerhard F. Ecker3, James E. Bradner1,4,*, Georg E. Winter2,*

1

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA

2

CeMM Research Center for Molecular Medicine of the Austrian Academy of Science,

Lazarettgasse 14, AKH Bt. 25.3, 1090 Vienna, Austria 3

University of Vienna, Department of Pharmaceutical Chemistry, Althanstrasse 14, 1090 Vienna,

Austria 4

Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA

#

These authors contributed equally to this work

* Corresponding author

Corresponding Author Contact Information James E. Bradner, M.D Novartis Institutes for Biomedical Research 181 Massachusetts Avenue Cambridge, MA 02139 617-871-8800 [email protected]

Georg E. Winter, PhD CeMM Research Center for Molecular Medicine of the Austrian Academy of Science Lazarettgasse 14, AKH Bt. 25.3 Vienna, Austria +43-1-40160-70031 [email protected]

ACS Paragon Plus Environment

1

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 2 of 21

2

Page 3 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Abstract Protein degradation is an emerging therapeutic strategy with a unique molecular pharmacology that enables the disruption of all functions associated with a target. This is particularly relevant for proteins depending on molecular scaffolding, such as transcription factors or receptor tyrosine kinases (RTKs). To address tractability of multiple RTKs for chemical degradation by the E3 ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN), we synthesized a series of phthalimide degraders based on the promiscuous kinase inhibitors sunitinib and PHA665752. While both series failed to induce degradation of their consensus targets, individual molecules displayed pronounced efficacy in leukemia cell lines. Orthogonal target identification supported by molecular docking led us to identify the translation termination factor G1 to S phase transition 1 (GSPT1) as a converging off-target resulting from inadvertent E3 ligase modulation. This research highlights the importance of monitoring degradation events that are independent of the respective targeting ligand as a unique feature of small-molecule degraders.


3


Page 4 of 21

Precision medicine aims to understand a pathologic condition, and tailor targeted therapeutic interventions for individual pharmacologic interference. This strategy depends on well-understood and precise drugs, but rationally designed agents often inhibit multiple protein targets. Poly-pharmacologic features are observed with histone deacetylase inhibitors,1 Poly (ADP-ribose) polymerase (PARP) inhibitors,2 and are particularly well-documented for kinase inhibitors.3-6 Ligand-induced targeted protein degradation has recently emerged as a novel paradigm in drug discovery. Small molecule degraders hijack the ubiquitin-proteasome system (UPS) to eliminate a protein of interest (POI). Molecularly, this is achieved using heterobifunctional molecules that induce proximity between a POI and an E3 ligase, leading to POI ubiquitination and degradation. Immunomodulatory drugs (IMiDs) bind the substrate adaptor cereblon (CRBN) of the CRL4CRBN E3 ligase, and inspired by their mechanism of action,7-9 we recently set out to hijack CRL4CRBN for the first in vivo target protein degradation via a modular, all-small molecule strategy.10,

11

We achieved this by

conjugation of a phthalimide moiety to a competitive antagonist of bromo- and extraterminal domain (BET) proteins, resulting in a selective BET degrader.10 Complementary approaches harnessed the Von Hippel-Lindau (VHL) E3 ligase to degrade the estrogenrelated receptor alpha (ERRα),12 the receptor interacting serine/threonine kinase 2 (RIPK2)12, the TANK binding kinase 1 (TBK1),13 or BET bromodomains,14 providing evidence for extensibility, both on the target and the E3 ligase. Target protein degradation results in a holistic perturbation as it allows chemical disruption of protein functions outside of the addressable binding pocket. It features non-traditional pharmacologic characteristics such as non-stoichiometric target turnover without drug turnover.12 Moreover, choice of E3 ligase and linker design are unexpected


4

Page 5 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


means to engineer selectivity into promiscuous targeting ligands.14-20 However, less attention has been brought to off-target effects caused by non-obvious modulation of the E3 ligase receptor. Here, we initially set out to map the amenability of multiple RTKs for targeted protein degradation by synthesizing phthalimide conjugates based on the non-selective kinase inhibitors sunitinib and PHA665752. Unexpectedly, both series failed to induce degradation of their consensus targets, while individual molecules displayed pronounced anti-proliferative effects in acute leukemia cell lines. Deploying orthogonal proteomics strategies, we identified that the most potent molecules act independent of their respective targeting ligand, and cause destabilization of the translation termination factor GSPT1 by serendipitous CRL4CRBN modulation. Subsequent molecular modeling provided structural evidence for the observed selectivity among individual degraders. Collectively, this study contributes to an improved understanding of the novel pharmacologic principles of targeted protein degradation. To generate kinase degraders, we conjugated sunitinib21 and PHA66575222 to CRL4CRBN E3 ligase-binding ligands based on phthalimide SAR. We exploited the solvent exposed amine groups of the kinase inhibitors for chemical derivatization,23,

24

and attached the

phthalimide moiety through various linkers at both the 4- and 5-position of the phthalimide (Supplementary Figure 1a, Supplementary Tables 1,2). All compounds were assessed for destabilization of their consensus RTK target, and their efficacy in RTKaddicted cell lines. To that end, PHA665752-based degraders (compounds 1-11) were assayed for their capacity to degrade the hepatocyte growth factor receptor (c-MET) in the squamous lung carcinoma cell line EBC-1. We did not observe significant changes in c-MET protein levels following incubation with either molecule at 10 µM (Supplementary


5


Page 6 of 21

Figure 1b). In line with this, none of the assayed molecules were comparably efficacious to the parental molecule PHA665752 in viability assays, (Supplementary Figure 1c). Similarly, the series of sunitinib-based degraders (compounds 12-26) failed to induce destabilization of the KIT proto-oncogene receptor tyrosine kinase (c-KIT) in GIST-T1 cells (Figure 1a), and failed to exceed sunitinib efficacy in cell viability studies (Figure 1b). Notably, representative phthalimide conjugates conserved binding to c-KIT or c-MET in the low nanomolar range (Supplementary Figure 1d).

However, when we assayed

compounds 12-26 in Kasumi-1 cells, a c-KIT dependent acute myeloid leukemia (AML) cell line, we observed that a single phthalimide conjugate, 22 (MI-389), featured an antiproliferative effect (EC50=21.3 nM) comparable to the cellular potency of sunitinib (EC50 = 17.3 nM) (Figure 1b, c). Surprisingly, MI-389 treatment failed to prompt c-KIT degradation, arguing for an alternative mechanism of action (Supplementary Figure 1e). Consistently, MI-389 outperformed sunitinib in three additional AML (HL60, MV4;11, NB4), as well as T-cell acute lymphobastic leukemia (T-ALL) cell lines (CCRF-CEM, JURKAT, MOLT4) that all are c-KIT independent (Figure 1d). An alternative mechanism of action was further supported by differential induction of apoptosis when comparing sunitinib and MI-389 (Figure 1e). This was further corroborated by comparative testing of drug impact on global gene activity via mRNA sequencing. While six hour sunitinib treatment only caused modest transcriptional impact, the effect of MI-389 differed qualitatively as well as quantitatively compared to sunitinib (Figure 1f, Supplementary Figure 1f, Supplementary Table 3, p2-fold) and significant (p

Translation Termination Factor GSPT1 Is a Phenotypically Relevant

Recommend Documents