Targeted Degradation of MDM2 as a New Approach to Improve the

Dec 21, 2018 - MDM2 is a key oncogenic protein that serves as a negative regulator of the tumor suppressor p53. While a number of inhibitors of the ...
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Viewpoint Cite This: J. Med. Chem. 2019, 62, 445−447

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Targeted Degradation of MDM2 as a New Approach to Improve the Efficacy of MDM2-p53 Inhibitors Ryan P. Wurz* and Victor J. Cee

J. Med. Chem. 2019.62:445-447. Downloaded from pubs.acs.org by 91.243.190.156 on 01/25/19. For personal use only.

Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States ABSTRACT: MDM2 is a key oncogenic protein that serves as a negative regulator of the tumor suppressor p53. While a number of inhibitors of the MDM2-p53 interaction have progressed to clinical testing as treatments for a variety of hematologic and solid tumor cancers, the results thus far have been mixed, with perhaps the strongest responses observed in relapsed/ refractory acute myeloid leukemia (AML). In an effort to improve the efficacy for this class of compounds, researchers have turned to targeted degradation of MDM2. IMiD-based MDM2 PROTAC 8, which potently reduces MDM2 protein levels through targeted degradation, exhibits enhanced efficacy in the RS4;11 xenograft model relative to a nondegrading MDM2-p53 inhibitor MI-1061.

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he transcription factor p53 plays a pivotal role in apoptosis of cancer cells, and its inactivation is a major contributing factor in tumorigenesis. The numerous functions of p53 are regulated by murine double minute 2 (MDM2). MDM2 directly binds to and blocks the N-terminal transcriptional activation domain of p53, promotes export of p53 from the nucleus to the cytoplasm, and induces degradation of p53 via ubiquitination through its E3 ligase activity. One mechanism of p53 inactivation in human cancers is the overexpression of MDM2, and the disruption of the MDM2p53 protein−protein interaction with small molecules has been viewed as an attractive strategy for boosting p53 levels in cancers, thereby promoting apoptosis. Several small-molecule inhibitors of the MDM2-p53 interaction have entered into clinical trials for the treatment of multiple cancer types (Figure 1) but have thus far shown mixed response rates. Doses have been limited by hematological and related toxicities including neutropenia, febrile neutropenia, prolonged cytopenia and bone marrow failure, as well as gastrointestinal toxicity.1−10

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TARGETED DEGRADATION OF MDM2 USING IMID-DERIVED PROTACS Targeted degradation has created new opportunities for interdiction of previously “undruggable” or ineffectively drugged targets through the creation of heterobifunctional molecules that tether a ligand that binds a target protein and a ligand that binds an E3 ubiquitin ligase.11 These chimeric molecules, occasionally called PROTACs (PROteolysis TArgeting Chimeras), facilitate complex formation of the target protein with an E3 ligase, resulting in ubiquitination of the target protein and its subsequent degradation by the 26S proteasome. Pharmacological outcomes arising from protein elimination by the proteasome could greatly contrast to those obtained by reversible or covalent inhibition, especially if the target protein has scaffolding functions or a low resynthesis rate. Furthermore, it has been shown that PROTACs with picomolar activity12 can be designed from binding partners of modest affinity due to both cooperative complex formation and the catalytic nature of these bispecific molecules. Therefore, pursuing a targeted degradation approach to interdict the © 2018 American Chemical Society

Figure 1. Six representative MDM2-p53 inhibitors that have entered human clinical trials.

Received: December 12, 2018 Published: December 21, 2018 445

DOI: 10.1021/acs.jmedchem.8b01945 J. Med. Chem. 2019, 62, 445−447

Journal of Medicinal Chemistry

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depletion evident as early as 3 h and near complete depletion by 24 h. The pharmacokinetic profile of 8 (not provided), would be required to understand the pharmacokinetic/ pharmacodynamic (PKPD) relationship. Multiple iv-dosing of 8 at 25 mg/kg every second day (Q2D) in the RS4;11 showed up to 50% tumor regression with no significant weight loss or other signs of toxicity. These results compared favorably to oral dosing at 100 mg/kg (5 days/week via oral gavage) of 7 which resulted in only modest tumor growth inhibition (∼50% TGI) in this model. In conclusion, IMiD-MDM2 PROTAC 8 effectively degrades MDM2 in vitro and in vivo, and when dosed iv at 25 mg/kg on a Q2D schedule, it appears to have significantly improved efficacy relative to a typical MDM2-p53 inhibitor, without any signs of toxicity. As small molecule MDM2-p53 inhibitors are already associated with clinical dose-limiting toxicities that are believed to be due to on-mechanism activation of p53 in normal cells and tissues such as bone marrow, spleen, and small intestines, it remains an open question whether the PROTAC approach reported by Li et al. will lead to improved efficacy without substantially increasing toxicity in human cancer patients.

MDM2-p53 protein−protein interaction could result in improved potency and sustained pharmacological effect substantially different from traditional inhibitors. Li et al. at the University of Michigan have recently disclosed the structures of potent MDM2 degraders.13 Using MDM2p53 inhibitor 7 (MI-1061) and IMiDs as the ligase binders (which target the CRL4-CRBN E3 ubiquitin ligase often referred to as cereblon), it was possible to obtain potent degraders of MDM2 after careful linker optimization (8, Figure 2). Control experiments confirmed that MDM2 degradation

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1 (805) 313 5400. ORCID

Ryan P. Wurz: 0000-0003-1413-5208

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ACKNOWLEDGMENTS The authors would like thank John Allen, Rati Verma, and Jude Canon for helpful suggestions in the editing of this Viewpoint.

Figure 2. Structures of MDM2-p53 inhibitor 7 (MI-1061) and IMiDMDM2 PROTAC 8 (MD-224).

was “on-mechanism” as cotreatment with lenalidomide, a cereblon binder, effectively blocked MDM2 degradation via competitive displacement of cereblon from the ternary complex. Preincubation with proteasome inhibitors MG-132 and PR-171 or a neddylation inhibitor MLN4924 also blocked MDM2 degradation induced by 8, further supporting the targeted degradation mechanism. Compound 8 is >10−50 times more potent than the parent MDM2 inhibitor 7 in induction of p53 activation and in inhibition of cell growth in the RS4;11 and MV4;11 cell lines. Compared to 8 in RS4;11 cells, MDM2-p53 reversible inhibitor 7 showed an increase in MDM2 protein levels as observed by Western blot analysis in addition to the desired increase in free p53 protein. Transcriptional upregulation of MDM2, the cell cycle regulator gene p21, and pro-apoptotic PUMA but not TP53, the gene encoding p53, was observed at 6 h by qRT-PCR analysis. IMiD-MDM2 PROTAC 8 also led to transcriptional gene upregulation of MDM2, but in contrast to 7, MDM2 protein was effectively depleted. MDM2 is itself an ubiquitin ligase and one unanswered question is whether CRL4-CRBN levels were impacted by MDM2-induced ubiquitination and subsequent proteasomal degradation. Compound 8 exhibited low nanomolar inhibitory activity in cell growth across a panel of AML cell lines suggesting that this MDM2 degradation strategy could be broadly applied in AML. Two AML cell lines bearing inactivating mutations in p53 were unresponsive to compound 8 as expected. In the RS4;11 xenograft model, a single 25 mg/kg iv dose of 8 exhibited a time-dependent reduction in MDM2 protein levels, with some

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DOI: 10.1021/acs.jmedchem.8b01945 J. Med. Chem. 2019, 62, 445−447