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1.
ACS Chem Biol ; 19(2): 442-450, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38305738

RESUMO

Targeted protein degradation with proteolysis targeting chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing the SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets.


Assuntos
Proteínas Culina , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/metabolismo , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
2.
ACS Chem Biol ; 19(1): 22-36, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38150587

RESUMO

The papain-like protease of SARS-COV-2 is essential for viral replication and pathogenesis. Its location within a much larger multifunctional protein, NSP3, makes it an ideal candidate for a targeted degradation approach capable of eliminating multiple functions with a single-molecule treatment. In this work, we have developed a HiBiT-based cellular model to study NSP3 degradation and used this platform for the discovery of monovalent NSP3 degraders. We present previously unreported degradation activity of published papain-like protease inhibitors. Follow-up exploration of structure-activity relationships and mechanism-of-action studies points to the recruitment of the ubiquitin-proteasome machinery that is solely driven by site occupancy, regardless of molecular features of the ligand. Supported by HDX data, we hypothesize that binding-induced structural changes in NSP3 trigger the recruitment of an E3 ligase and lead to proteasomal degradation.


Assuntos
COVID-19 , Proteases Semelhantes à Papaína de Coronavírus , Papaína , Humanos , Papaína/metabolismo , Proteínas não Estruturais Virais/metabolismo , SARS-CoV-2/química , Inibidores de Proteases/metabolismo
3.
bioRxiv ; 2023 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-37961622

RESUMO

ß-catenin (CTNNB1) is an oncogenic transcription factor that is important in cell-cell adhesion and transcription of cell proliferation and survival genes that drives the pathogenesis of many different types of cancers. However, direct pharmacological targeting of CTNNB1 has remained challenging deeming this transcription factor as "undruggable." Here, we have performed a screen with a library of cysteine-reactive covalent ligands to identify a monovalent degrader EN83 that depletes CTNNB1 in a ubiquitin-proteasome-dependent manner. We show that EN83 directly and covalently targets CTNNB1 through targeting four distinct cysteines within the armadillo repeat domain-C439, C466, C520, and C619-leading to a destabilization of CTNNB1. Using covalent chemoproteomic approaches, we show that EN83 directly engages CTNNB1 in cells with a moderate degree of selectivity. We further demonstrate that direct covalent targeting of three of these four cysteines--C466, C520, and C619--in cells contributes to CTNNB1 degradation in cells. We also demonstrate that EN83 can be further optimized to yield more potent CTNNB1 binders and degraders. Our results show that chemoproteomic approaches can be used to covalently target and degrade challenging transcription factors like CTNNB1 through a destabilization-mediated degradation.

4.
bioRxiv ; 2023 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-37904950

RESUMO

Targeted protein degradation with Proteolysis Targeting Chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets.

5.
Am J Hematol ; 98(3): 449-463, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36594167

RESUMO

The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.


Assuntos
Imunoconjugados , Linfoma não Hodgkin , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/patologia , Rituximab/uso terapêutico , Imunoconjugados/uso terapêutico
6.
Nat Chem Biol ; 19(1): 55-63, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36577875

RESUMO

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.


Assuntos
Proteínas Nucleares , Fatores de Transcrição , Proteínas Nucleares/metabolismo , Microscopia Crioeletrônica , Fatores de Transcrição/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ligases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
7.
Science ; 378(6619): 549-553, 2022 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-36378961

RESUMO

Cereblon (CRBN) is a ubiquitin ligase (E3) substrate receptor protein co-opted by CRBN E3 ligase modulatory drug (CELMoD) agents that target therapeutically relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBN's thalidomide-binding domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We performed cryo-electron microscopy analyses of the DNA damage-binding protein 1 (DDB1)-CRBN apo complex and compared these structures with DDB1-CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an open conformation to the canonical closed conformation. The neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy and informing structure-guided design strategies to improve therapeutic efficacy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ubiquitina-Proteína Ligases , Proteínas Adaptadoras de Transdução de Sinal/química , Microscopia Crioeletrônica , Talidomida/química , Ubiquitina-Proteína Ligases/química , Domínios Proteicos , Regulação Alostérica
8.
Ann N Y Acad Sci ; 1510(1): 79-99, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35000205

RESUMO

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.


Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Autofagia/fisiologia , Humanos , Organelas , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteólise , Ubiquitina/metabolismo
9.
Nat Biotechnol ; 40(1): 86-93, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34531539

RESUMO

Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq). TraCe-seq is a method that captures at clonal resolution the origin, fate and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual epidermal growth factor receptor (EGFR) inhibitor-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. We identified a loss of antigrowth activity associated with targeted degradation of EGFR protein and an essential role of the endoplasmic reticulum (ER) protein processing pathway in anti-EGFR therapeutic efficacy. Our results suggest that targeted degradation is not always superior to enzymatic inhibition and establish TraCe-seq as an approach to study how preexisting transcriptional programs affect treatment responses.


Assuntos
Antineoplásicos , Neoplasias Pulmonares , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Análise de Célula Única/métodos
10.
Nat Commun ; 12(1): 4608, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326324

RESUMO

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.


Assuntos
Anticorpos/química , Peptídeos/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Células Cultivadas , Cristalografia por Raios X/métodos , Humanos , Espectrometria de Massas/métodos , Ligação Proteica , Proteômica/métodos , Coelhos , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/imunologia , Ubiquitinação
11.
J Med Chem ; 64(16): 11841-11856, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34251202

RESUMO

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Carbolinas/uso terapêutico , Antagonistas do Receptor de Estrogênio/uso terapêutico , Receptor alfa de Estrogênio/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Carbolinas/química , Carbolinas/farmacocinética , Cães , Antagonistas do Receptor de Estrogênio/química , Antagonistas do Receptor de Estrogênio/farmacocinética , Feminino , Humanos , Células MCF-7 , Macaca fascicularis , Camundongos , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Cell Death Differ ; 28(2): 591-605, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33432113

RESUMO

The ubiquitin system is complex, multifaceted, and is crucial for the modulation of a vast number of cellular processes. Ubiquitination is tightly regulated at different levels by a range of enzymes including E1s, E2s, and E3s, and an array of DUBs. The UPS directs protein degradation through the proteasome, and regulates a wide array of cellular processes including transcription and epigenetic factors as well as key oncoproteins. Ubiquitination is key to the dynamic regulation of programmed cell death. Notably, the TNF signaling pathway is controlled by competing ubiquitin conjugation and deubiquitination, which governs both proteasomal degradation and signaling complex formation. In the inflammatory response, ubiquitination is capable of both activating and dampening inflammasome activation through the control of either protein stability, complex formation, or, in some cases, directly affecting receptor activity. In this review, we discuss the enzymes and targets in the ubiquitin system that regulate fundamental cellular processes regulating cell death, and inflammation, as well as disease consequences resulting from their dysregulation. Finally, we highlight several pre-clinical and clinical compounds that regulate ubiquitin system enzymes, with the aim of restoring homeostasis and ameliorating diseases.


Assuntos
Inflamação/metabolismo , Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Apoptose , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo
14.
Mol Cell ; 77(5): 1092-1106.e9, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31973889

RESUMO

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.


Assuntos
Cromatografia Líquida de Alta Pressão , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Células HEK293 , Humanos , Cinética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Mapas de Interação de Proteínas , Proteólise , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética
15.
Bioorg Med Chem Lett ; 30(4): 126907, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31902710

RESUMO

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.


Assuntos
Anticorpos Monoclonais/imunologia , Portadores de Fármacos/química , Receptor alfa de Estrogênio/imunologia , Anticorpos Monoclonais/química , Antineoplásicos/química , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Desenho de Fármacos , Receptor alfa de Estrogênio/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Células MCF-7 , Proteólise/efeitos dos fármacos , Receptor ErbB-2/metabolismo
16.
Cell ; 178(4): 949-963.e18, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31353221

RESUMO

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.


Assuntos
Neoplasias da Mama/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Cinamatos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Antagonistas do Receptor de Estrogênio/uso terapêutico , Feminino , Fulvestranto/uso terapêutico , Células HEK293 , Xenoenxertos , Humanos , Indazóis/farmacologia , Ligantes , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Polimorfismo de Nucleotídeo Único , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
17.
Drug Discov Today Technol ; 31: 109-123, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31200854

RESUMO

The Ubiquitin/Proteasome System comprises an essential cellular mechanism for regulated protein degradation. Ubiquitination may also promote the assembly of protein complexes that initiate intracellular signaling cascades. Thus, proper regulation of substrate protein ubiquitination is essential for maintaining normal cellular physiology. Deubiquitinases are the class of enzymes responsible for removing ubiquitin modifications from target proteins and have been implicated in regulating human disease. As such, deubiquitinases are now recognized as emerging drug targets. Small molecule deubiquitinase inhibitors have been developed; among those, inhibitors for the deubiquitinases USP7 and USP14 are the best-characterized given that they are structurally validated. In this review we discuss the normal physiological roles of the USP7 and USP14 deubiquitinases as well as the pathological conditions associated with their dysfunction, with a focus on oncology and neurodegenerative diseases. We also review structural biology of USP7 and USP14 enzymes and the characterization of their respective inhibitors, highlighting the various molecular mechanisms by which these deubiquitinases may be functionally inhibited. Finally, we summarize the cellular and in vivo studies performed using the structurally-validated USP7 and USP14 inhibitors.


Assuntos
Pirróis/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Animais , Humanos , Estrutura Molecular , Pirróis/química , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo
19.
Cell Chem Biol ; 26(2): 156-177, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30554913

RESUMO

The ubiquitin/proteasome system is a primary conduit for selective intracellular protein degradation. Since its discovery over 30 years ago, this highly regulated system continues to be an active research area for drug discovery that is exemplified by several approved drugs. Here we review compounds in preclinical testing, clinical trials, and approved drugs, with the aim of highlighting innovative discoveries and breakthrough therapies that target the ubiquitin system.


Assuntos
Inibidores Enzimáticos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
20.
Nat Commun ; 9(1): 1162, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563501

RESUMO

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.


Assuntos
Enzimas Desubiquitinantes/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Proteína de Replicação A/metabolismo , Coloração e Rotulagem/métodos , Biocatálise , Domínio Catalítico , Cisteína/química , Cisteína/metabolismo , Enzimas Desubiquitinantes/classificação , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/química , Lisina/metabolismo , Sondas Moleculares , Proteína de Replicação A/genética , Sumoilação , Ubiquitinação
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