RESUMO
Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Membrana , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bioensaio , Citosol , Imunidade Inata , Ligantes , Proteínas de Membrana/metabolismoRESUMO
Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.
Assuntos
Fibrose Cística , Quimera/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/uso terapêutico , Humanos , Ligantes , Ubiquitina/metabolismoRESUMO
While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Cisteína , Antivirais/farmacologia , Antivirais/química , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Simulação de Acoplamento MolecularRESUMO
Proteolysis-targeting chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting C186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications by demonstrating that a PROTAC linking EN106 to the BET bromodomain inhibitor JQ1 or the kinase inhibitor dasatinib leads to the degradation of BRD4 and BCR-ABL, respectively. Our study showcases a covalent ligand that targets a natural E3 ligase-substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters suitable for TPD applications.
Assuntos
Acetamidas/química , Proteínas de Ciclo Celular/metabolismo , Proteólise , Complexos Ubiquitina-Proteína Ligase/metabolismo , Animais , Azepinas/química , Sítios de Ligação , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cisteína/química , Dasatinibe/química , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Triazóis/química , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.
Assuntos
Neoplasias da Mama , Metionina , Humanos , Feminino , Quinase 4 Dependente de Ciclina/metabolismo , Ligantes , Fosforilação , Oxirredução , Racemetionina/metabolismoRESUMO
Molecular glues are an intriguing therapeutic modality that harness small molecules to induce interactions between proteins that typically do not interact. However, such molecules are rare and have been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention. We postulated that natural products bearing one or more electrophilic sites may be an unexplored source of new molecular glues, potentially acting through multicovalent attachment. Using chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells, and engage in molecular glue interactions with the neosubstrate tumor-suppressor TP53, leading to p53 transcriptional activation and cell death. Our results reveal an anticancer mechanism of this natural product family, and highlight the potential for combining chemoproteomics and multicovalent natural products for the discovery of new molecular glues.
Assuntos
Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Polienos/química , Policetídeos/química , Alcamidas Poli-Insaturadas/química , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Descoberta de Drogas , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Conformação Molecular , Estrutura Molecular , Polienos/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Eletricidade Estática , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Leucemia Mieloide Aguda/metabolismo , Precursores de RNA/metabolismo , Sarcoma de Ewing/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Fator de Especificidade de Clivagem e Poliadenilação/genética , Células HEK293 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Piperazinas/farmacologia , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Sarcoma de Ewing/tratamento farmacológicoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Limoninas/farmacologia , Proteólise/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Limoninas/química , Limoninas/isolamento & purificação , Ubiquitina-Proteína LigasesRESUMO
The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
Assuntos
Proteínas de Transporte de Cátions/genética , Estresse do Retículo Endoplasmático/fisiologia , Receptor Notch1/genética , Animais , Apoptose , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/fisiologia , Linhagem Celular , Transformação Celular Neoplásica , Retículo Endoplasmático/fisiologia , Humanos , Mutação , Transporte Proteico , Receptor Notch1/fisiologia , Transdução de Sinais , Zinco/metabolismoRESUMO
YAP signaling pathway plays critical roles in tissue homeostasis, and aberrant activation of YAP signaling has been implicated in cancers. To identify tractable targets of YAP pathway, we have performed a pathway-based pooled CRISPR screen and identified tankyrase and its associated E3 ligase RNF146 as positive regulators of YAP signaling. Genetic ablation or pharmacological inhibition of tankyrase prominently suppresses YAP activity and YAP target gene expression. Using a proteomic approach, we have identified angiomotin family proteins, which are known negative regulators of YAP signaling, as novel tankyrase substrates. Inhibition of tankyrase or depletion of RNF146 stabilizes angiomotins. Angiomotins physically interact with tankyrase through a highly conserved motif at their N terminus, and mutation of this motif leads to their stabilization. Tankyrase inhibitor-induced stabilization of angiomotins reduces YAP nuclear translocation and decreases downstream YAP signaling. We have further shown that knock-out of YAP sensitizes non-small cell lung cancer to EGFR inhibitor Erlotinib. Tankyrase inhibitor, but not porcupine inhibitor, which blocks Wnt secretion, enhances growth inhibitory activity of Erlotinib. This activity is mediated by YAP inhibition and not Wnt/ß-catenin inhibition. Our data suggest that tankyrase inhibition could serve as a novel strategy to suppress YAP signaling for combinatorial targeted therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Receptores ErbB/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/antagonistas & inibidores , Tanquirases/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Angiomotinas , Antineoplásicos/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Regulação para Baixo , Cloridrato de Erlotinib/farmacologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas dos Microfilamentos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Tanquirases/química , Tanquirases/genética , Fatores de Transcrição , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Sinalização YAPRESUMO
A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.
Assuntos
Produtos Biológicos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Ascomicetos/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HCT116 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Polimorfismo de Nucleotídeo Único/genética , Canais de Translocação SEC , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.
Assuntos
Processamento Alternativo , Atrofia Muscular Espinal/tratamento farmacológico , RNA de Cadeia Dupla/agonistas , Ribonucleoproteína Nuclear Pequena U1/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Sítios de Ligação , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/mortalidade , Atrofia Muscular Espinal/patologia , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteólise , Precursores de RNA/agonistas , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/metabolismo , Análise de Sobrevida , Proteína 2 de Sobrevivência do Neurônio Motor/química , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Identification and validation of drug-resistant mutations can provide important insights into the mechanism of action of a compound. Here we demonstrate the feasibility of such an approach in mammalian cells using next-generation sequencing of drug-resistant clones and CRISPR-Cas9-mediated gene editing on two drug-target pairs, 6-thioguanine-HPRT1 and triptolide-ERCC3. We showed that disrupting functional HPRT1 allele or introducing ERCC3 point mutations by gene editing can confer drug resistance in cells.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Linhagem Celular/efeitos dos fármacos , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Diterpenos/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Compostos de Epóxi/farmacologia , Células HCT116 , Humanos , Hipoxantina Fosforribosiltransferase/genética , Mamíferos , Fenantrenos/farmacologia , Mutação Puntual , Reprodutibilidade dos Testes , Tioguanina/farmacologiaRESUMO
Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.
Assuntos
Proteínas Hedgehog/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Cromatografia de Afinidade , Proteômica , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.
Assuntos
Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tanquirases/antagonistas & inibidores , Proteínas Wnt/antagonistas & inibidores , Proteína Axina , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteômica , Proteínas Repressoras/química , Tanquirases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action.
Assuntos
Antivirais , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Animais , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Cães , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Células Jurkat , Infecções por Vírus Respiratório Sincicial/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Linfócitos T/virologia , Células VeroRESUMO
Ophiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anticancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of the OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets: lysine-72 of cytochrome c oxidase subunit 5A (COX5A) and cysteine-53 of mitochondrial hypoxia induced gene 1 domain family member 2A (HIGD2A). These two subunit proteins are part of complex IV (cytochrome C oxidase) within the electron transport chain and contributed significantly to the antiproliferative activity of OPA. OPA activated mitochondrial respiration in a COX5A- and HIGD2A-dependent manner, leading to an initial spike in mitochondrial ATP and heightened mitochondrial oxidative stress. OPA compromised mitochondrial membrane potential, ultimately leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anticancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Mitocôndrias , Sesterterpenos , Humanos , Sesterterpenos/farmacologia , Sesterterpenos/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Estresse Oxidativo/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.