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1.
Cell ; 183(6): 1714-1731.e10, 2020 12 10.
Article in English | MEDLINE | ID: mdl-33275901

ABSTRACT

Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.


Subject(s)
Protein Kinases/metabolism , Proteolysis , Proteome/metabolism , Adult , Cell Line , Databases, Protein , Female , Humans , Male , Middle Aged , Proteasome Endopeptidase Complex/metabolism , Protein Kinases/genetics , Proteomics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ubiquitin-Protein Ligases/metabolism , Young Adult
2.
Chemistry ; : e202400430, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38818652

ABSTRACT

BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other hematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.

3.
Nat Commun ; 15(1): 2743, 2024 Mar 29.
Article in English | MEDLINE | ID: mdl-38548768

ABSTRACT

Overexpression of BCL-xL and BCL-2 play key roles in tumorigenesis and cancer drug resistance. Advances in PROTAC technology facilitated recent development of the first BCL-xL/BCL-2 dual degrader, 753b, a VHL-based degrader with improved potency and reduced toxicity compared to previous small molecule inhibitors. Here, we determine crystal structures of VHL/753b/BCL-xL and VHL/753b/BCL-2 ternary complexes. The two ternary complexes exhibit markedly different architectures that are accompanied by distinct networks of interactions at the VHL/753b-linker/target interfaces. The importance of these interfacial contacts is validated via functional analysis and informed subsequent rational and structure-guided design focused on the 753b linker and BCL-2/BCL-xL warhead. This results in the design of a degrader, WH244, with enhanced potency to degrade BCL-xL/BCL-2 in cells. Using biophysical assays followed by in cell activities, we are able to explain the enhanced target degradation of BCL-xL/BCL-2 in cells. Most PROTACs are empirically designed and lack structural studies, making it challenging to understand their modes of action and specificity. Our work presents a streamlined approach that combines rational design and structure-based insights backed with cell-based studies to develop effective PROTAC-based cancer therapeutics.


Subject(s)
Neoplasms , Proto-Oncogene Proteins c-bcl-2 , Humans , bcl-X Protein/metabolism
4.
Cell Chem Biol ; 30(2): 203-213.e17, 2023 02 16.
Article in English | MEDLINE | ID: mdl-36750097

ABSTRACT

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS-032, a CDK9 inhibitor. The lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome-dependent manner and is much more potent than SNS-032 against various tumor cells in vitro. Mechanistically, we identified KEAP1 as the E3 ligase recruited by 955 to degrade CDK9 through a TurboID-based proteomics study, which was further confirmed by KEAP1 knockout and the nanoBRET ternary complex formation assay. In addition, PL-ceritinib conjugate can degrade EML4-ALK fusion oncoprotein, suggesting that PL may have a broader application as a covalent E3 ligase ligand in targeted protein degradation.


Subject(s)
NF-E2-Related Factor 2 , Ubiquitin-Protein Ligases , Proteolysis , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , Ubiquitin-Protein Ligases/metabolism , Ligands
5.
Nat Commun ; 14(1): 686, 2023 02 08.
Article in English | MEDLINE | ID: mdl-36754960

ABSTRACT

Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.


Subject(s)
Endopeptidases , Ubiquitin , Ubiquitin/metabolism , Endopeptidases/metabolism , Peptide Hydrolases/metabolism , Structure-Activity Relationship , Deubiquitinating Enzymes/metabolism , Ubiquitination
6.
J Hematol Oncol ; 15(1): 23, 2022 03 09.
Article in English | MEDLINE | ID: mdl-35260176

ABSTRACT

KRAS mutations are the most common oncogenic drivers. Sotorasib (AMG510), a covalent inhibitor of KRASG12C, was recently approved for the treatment of KRASG12C-mutated non-small cell lung cancer (NSCLC). However, the efficacy of sotorasib and other KRASG12C inhibitors is limited by intrinsic resistance in colorectal cancer (CRC) and by the rapid emergence of acquired resistance in all treated tumors. Therefore, there is an urgent need to develop novel combination therapies to overcome sotorasib resistance and to maximize its efficacy. We assessed the effect of sotorasib alone or in combination with DT2216 (a clinical-stage BCL-XL proteolysis targeting chimera [PROTAC]) on KRASG12C-mutated NSCLC, CRC and pancreatic cancer (PC) cell lines using MTS cell viability, colony formation and Annexin-V/PI apoptosis assays. Furthermore, the therapeutic efficacy of sotorasib alone and in combination with DT2216 was evaluated in vivo using different tumor xenograft models. We observed heterogeneous responses to sotorasib alone, whereas its combination with DT2216 strongly inhibited viability of KRASG12C tumor cell lines that partially responded to sotorasib treatment. Mechanistically, sotorasib treatment led to stabilization of BIM and co-treatment with DT2216 inhibited sotorasib-induced BCL-XL/BIM interaction leading to enhanced apoptosis in KRASG12C tumor cell lines. Furthermore, DT2216 co-treatment significantly improved the antitumor efficacy of sotorasib in vivo. Collectively, our findings suggest that due to cytostatic activity, the efficacy of sotorasib is limited, and therefore, its combination with a pro-apoptotic agent, i.e., DT2216, shows synergistic responses and can potentially overcome resistance.


Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , bcl-X Protein/genetics , bcl-X Protein/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , Piperazines , Proteolysis , Proto-Oncogene Proteins p21(ras)/genetics , Pyridines , Pyrimidines
7.
Cell Chem Biol ; 28(1): 78-87.e3, 2021 01 21.
Article in English | MEDLINE | ID: mdl-33007217

ABSTRACT

Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.


Subject(s)
Proteomics , Ubiquitin-Specific Peptidase 7/analysis , Biocatalysis , Cells, Cultured , Enzyme Inhibitors/pharmacology , Humans , Substrate Specificity , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitination
8.
J Med Chem ; 64(19): 14230-14246, 2021 10 14.
Article in English | MEDLINE | ID: mdl-34533954

ABSTRACT

BCL-XL and BCL-2 are important targets for cancer treatment. BCL-XL specific proteolysis-targeting chimeras (PROTACs) have been developed to circumvent the on-target platelet toxicity associated with BCL-XL inhibition. However, they have minimal effects on cancer cells that are dependent on BCL-2 or both BCL-XL and BCL-2. Here we report a new series of BCL-PROTACs. The lead PZ703b exhibits high potency in inducing BCL-XL degradation and in inhibiting but not degrading BCL-2, showing a hybrid dual-targeting mechanism of action that is unprecedented in a PROTAC molecule. As a result, PZ703b is highly potent in killing BCL-XL dependent, BCL-2 dependent, and BCL-XL/BCL-2 dual-dependent cells in an E3 ligase (VHL)-dependent fashion. We further found that PZ703b forms stable {BCL-2:PROTAC:VCB} ternary complexes in live cells that likely contribute to the enhanced BCL-2 inhibition by PZ703b. With further optimization, analogues of PZ703b could potentially be developed as effective antitumor agents by co-targeting BCL-XL and BCL-2.


Subject(s)
Drug Discovery , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , bcl-X Protein/antagonists & inhibitors , Dose-Response Relationship, Drug , Humans , Molecular Structure , Proteolysis/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Structure-Activity Relationship , bcl-X Protein/metabolism
9.
Nat Commun ; 12(1): 6896, 2021 11 25.
Article in English | MEDLINE | ID: mdl-34824248

ABSTRACT

PROteolysis-TArgeting Chimeras (PROTACs) have emerged as an innovative drug development platform. However, most PROTACs have been generated empirically because many determinants of PROTAC specificity and activity remain elusive. Through computational modelling of the entire NEDD8-VHL Cullin RING E3 ubiquitin ligase (CRLVHL)/PROTAC/BCL-xL/UbcH5B(E2)-Ub/RBX1 complex, we find that this complex can only ubiquitinate the lysines in a defined band region on BCL-xL. Using this approach to guide our development of a series of ABT263-derived and VHL-recruiting PROTACs, we generate a potent BCL-xL and BCL-2 (BCL-xL/2) dual degrader with significantly improved antitumor activity against BCL-xL/2-dependent leukemia cells. Our results provide experimental evidence that the accessibility of lysines on a target protein plays an important role in determining the selectivity and potency of a PROTAC in inducing protein degradation, which may serve as a conceptual framework to guide the future development of PROTACs.


Subject(s)
Antineoplastic Agents/pharmacology , Leukemia/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-X Protein/metabolism , Antineoplastic Agents/chemistry , Cell Line , Cell Survival/drug effects , Humans , Leukemia/drug therapy , Leukemia/genetics , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Models, Molecular , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Conformation , Proteolysis , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Von Hippel-Lindau Tumor Suppressor Protein/chemistry , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , bcl-X Protein/chemistry , bcl-X Protein/genetics
10.
Eur J Med Chem ; 192: 112186, 2020 Apr 15.
Article in English | MEDLINE | ID: mdl-32145645

ABSTRACT

Anti-apoptotic protein BCL-XL plays a key role in tumorigenesis and cancer chemotherapy resistance, rendering it an attractive target for cancer treatment. However, BCL-XL inhibitors such as ABT-263 cannot be safely used in the clinic because platelets solely depend on BCL-XL to maintain their viability. To reduce the on-target platelet toxicity associated with the inhibition of BCL-XL, we designed and synthesized PROTAC BCL-XL degraders that recruit CRBN or VHL E3 ligase because both of these enzymes are poorly expressed in human platelets compared to various cancer cell lines. We confirmed that platelet-toxic BCL-XL/2 dual inhibitor ABT-263 can be converted into platelet-sparing CRBN/VHL-based BCL-XL specific degraders. A number of BCL-XL degraders are more potent in killing cancer cells than their parent compound ABT-263. Specifically, XZ739, a CRBN-dependent BCL-XL degrader, is 20-fold more potent than ABT-263 against MOLT-4 T-ALL cells and has >100-fold selectivity for MOLT-4 cells over human platelets. Our findings further demonstrated the utility of PROTAC technology to achieve tissue selectivity through recruiting differentially expressed E3 ligases.


Subject(s)
Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , Blood Platelets/drug effects , Drug Discovery , Proteolysis/drug effects , Sulfonamides/pharmacology , bcl-X Protein/antagonists & inhibitors , Aniline Compounds/chemical synthesis , Aniline Compounds/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Tumor Cells, Cultured
11.
Sci Rep ; 10(1): 5324, 2020 03 24.
Article in English | MEDLINE | ID: mdl-32210275

ABSTRACT

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.


Subject(s)
Protease Inhibitors/pharmacology , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , MCF-7 Cells , Protease Inhibitors/chemistry , Ubiquitin-Specific Peptidase 7/chemistry , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitination/drug effects
12.
Cell Chem Biol ; 24(12): 1490-1500.e11, 2017 12 21.
Article in English | MEDLINE | ID: mdl-29056421

ABSTRACT

Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5-10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors.


Subject(s)
Protease Inhibitors/pharmacology , Thiophenes/pharmacology , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Catalytic Domain , Dose-Response Relationship, Drug , Drug Design , Humans , Models, Molecular , Molecular Structure , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Structure-Activity Relationship , Substrate Specificity , Thiophenes/chemistry , Ubiquitin/metabolism , Ubiquitin-Specific Peptidase 7/metabolism
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