RESUMO
WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.
Assuntos
Regulação Alostérica , Descoberta de Drogas , Inibidores Enzimáticos , Proteômica , Helicase da Síndrome de Werner , Animais , Feminino , Humanos , Masculino , Camundongos , Regulação Alostérica/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Cisteína/efeitos dos fármacos , Cisteína/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Instabilidade de Microssatélites , Modelos Moleculares , Helicase da Síndrome de Werner/antagonistas & inibidores , Helicase da Síndrome de Werner/química , Helicase da Síndrome de Werner/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Morte Celular/efeitos dos fármacos , Trifosfato de Adenosina/metabolismoRESUMO
The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.
Assuntos
Cisteína , Proteômica , Transdução de Sinais , Citocinas , Isoformas de ProteínasRESUMO
Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.
Assuntos
Complexos Ubiquitina-Proteína Ligase/fisiologia , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Receptores Androgênicos/metabolismo , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Reversible protein phosphorylation plays essential roles in coordinating cell division and many other biological processes. Cell cycle regulation by opposing kinase and protein phosphatase activities is often complex and major challenges exist in identifying the direct substrates of these enzymes and the specific sites at which they act. While cell cycle kinases are known to exhibit strict substrate specificities important for coordinating the complex events of cell division, phosphatases have only recently been recognized to exert similarly precise regulatory control over cell cycle events through timely dephosphorylation of specific substrates. The molecular determinants for substrate recognition by many phosphatases that function in cell division are still poorly delineated. To understand phosphatase specificity, it is critical to employ methods that monitor the dephosphorylation of individual phosphorylation sites on physiologically relevant substrates. Here, using the cell cycle phosphatase Cdc14 as an example, we describe two methods for studying phosphatase specificity, one using synthetic phosphopeptide substrates and the other using intact phosphoprotein substrates. These methods are useful for targeted characterization of small substrate sets and are also adaptable to large-scale applications for global specificity studies.
Assuntos
Ensaios Enzimáticos/métodos , Fosfoproteínas Fosfatases/metabolismo , Cromatografia Líquida , Espectrometria de Massas , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosforilação , Especificidade por SubstratoRESUMO
Faithful genome transmission during cell division requires precise, coordinated action of DNA metabolic enzymes, including proteins responsible for DNA damage detection and repair. Dynamic phosphorylation plays an important role in controlling repair enzymes during the DNA damage response (DDR). Cdc14 phosphatases oppose cyclin-dependent kinase (Cdk) phosphorylation and have been implicated in the DDR in several model systems. Here, we have refined the substrate specificity of budding yeast Cdc14 and, using this insight, identified the Holliday junction resolvase Yen1 as a DNA repair target of Cdc14. Cdc14 activation at anaphase triggers nuclear accumulation and enzymatic activation of Yen1, likely to resolve persistent recombinational repair intermediates. Consistent with this, expression of a phosphomimetic Yen1 mutant increased sister chromatid nondisjunction. In contrast, lack of Cdk phosphorylation resulted in constitutive activity and elevated crossover-associated repair. The precise timing of Yen1 activation, governed by core cell-cycle regulators, helps coordinate DNA repair with chromosome segregation and safeguards against genome destabilization.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Instabilidade Genômica , Resolvases de Junção Holliday/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Cromossomos Fúngicos , Quinases Ciclina-Dependentes/genética , Reparo do DNA , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Resolvases de Junção Holliday/genética , Mitose , Mutação , Fosforilação , Proteínas Tirosina Fosfatases/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Fatores de TempoRESUMO
The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1-5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient's Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ~15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.
Assuntos
Benzamidas/administração & dosagem , Técnicas Biossensoriais , Proteínas de Fusão bcr-abl/isolamento & purificação , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/administração & dosagem , Proteínas Tirosina Quinases/isolamento & purificação , Pirimidinas/administração & dosagem , Peptídeos Penetradores de Células/química , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Fosforilação , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismoRESUMO
Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Substituição de Aminoácidos , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Fosfatases de Especificidade Dupla/química , Fosfatases de Especificidade Dupla/genética , Humanos , Mutação de Sentido Incorreto , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação/fisiologia , Fosfosserina/química , Fosfosserina/metabolismo , Proteínas Tirosina Fosfatases , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Especificidade por Substrato/fisiologiaRESUMO
The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.
Assuntos
Proteínas de Bactérias/análise , Proteínas Fúngicas/análise , Espectrometria de Massas/métodos , Proteômica/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases/análise , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
Quantification of protein and PTM abundance in biological samples is an important component of proteomic studies. Label-free methods for quantification using MS are attractive because they are simple to implement and applicable to any experimental system. We demonstrate that PTM stoichiometry can be accurately measured using label-free quantification and selected reaction monitoring. Use of selected reaction monitoring is advantageous with complex biological samples and we show this approach can be used to quantify multiple PTMs independently on a single peptide.
Assuntos
Fosfopeptídeos/química , Proteínas/química , Sequência de Aminoácidos , Extratos Celulares , Proteínas Fúngicas/química , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Proteômica/métodos , Receptor EphA2/química , Proteínas Recombinantes/químicaRESUMO
MADS-domain proteins are important transcription factors involved in many biological processes of plants. Interactions between MADS-domain proteins are essential for their functions. In tomato (Solanum lycopersicum), the number of MIKC(c)-type MADS-domain proteins identified has totalled 36, but a large-scale interaction assay is lacking. In this study, 22 tomato MADS-domain proteins were selected from six functionally important subfamilies of the MADS-box gene family, to create the first large-scale tomato protein interaction network. Compared with Arabidopsis and petunia (Petunia hybrida), protein interaction patterns in tomato displayed both conservation and divergence. The majority of proteins that can be identified as putative orthologues exhibited conserved interaction patterns, and modifications were mostly found in genes underlining traits unique to tomato. JOINTLESS and RIN, characterized for their roles in abscission zone development and fruit ripening, respectively, showed enlarged interaction networks in comparison with their Arabidopsis and petunia counterparts. Novel interactions were also found for members of the expanded subfamilies, such as those represented by AP1/FUL and AP3/PI MADS-domain proteins. In search for higher order complexes, TM5 was found to be the preferred bridge among the five SEP-like proteins. Additionally, 16 proteins with the MADS-domain removed were used to assess the role of the MADS-domain in protein-protein interactions. The current work provides important knowledge for further functional and evolutionary study of the MADS-box genes in tomato.