Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 52
Filtrar
Mais filtros












Base de dados
Intervalo de ano de publicação
1.
bioRxiv ; 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38798327

RESUMO

Small molecule-mediated proteasomal degradation of proteins is a powerful tool for synthetic regulation of biological activity. To control Cas9 activity in cells, we engineered an anti-CRISPR protein, AcrIIA4, fused to a degradation (dTAG) or small molecule assisted shutoff (SMASh) tag. Co-expression of the tagged AcrIIA4 along with Cas9 and riboswitch-regulated sgRNAs enables precise tunable control of CRISPR activity by small molecule addition.

3.
Nature ; 628(8007): 442-449, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38538798

RESUMO

Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.


Assuntos
Complexos Multiproteicos , Mutação , Neoplasias , Proteína SMARCB1 , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Sistemas CRISPR-Cas , Edição de Genes , Neoplasias/genética , Neoplasias/metabolismo , Proteína SMARCB1/deficiência , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteólise , Ubiquitina/metabolismo
4.
Bioorg Med Chem ; 102: 117672, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38461554

RESUMO

Small molecules that chemically induce proximity between two proteins have been widely used to precisely modulate protein levels, stability, and activity. Recently, several studies developed novel strategies that employ heterobifunctional molecules that co-opt shuttling proteins to control the spatial localization of a target protein, unlocking new potential within this domain. Together, these studies lay the groundwork for novel targeted protein relocalization modalities that can rewire the protein circuitry and interactome to influence biological outcomes.


Assuntos
Imãs , Proteínas , Proteínas/química , Ligação Proteica
5.
PLoS Pathog ; 19(9): e1011169, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37669313

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposi's sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identify specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch.


Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/genética , Polifarmacologia , África , Cognição , Proteínas Serina-Treonina Quinases , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Serina-Treonina Quinases de Interação com Receptores
6.
Trends Pharmacol Sci ; 44(11): 750-752, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37758537

RESUMO

Achieving tumor-specific protein loss remains a challenge in the delivery of proteolysis-targeting chimeras (PROTACs) as cancer therapeutics. As a solution, Wang et al. recently developed nanoformulated PROTACs (NAPs), a novel photoactivatable degradation approach. This innovative class of compounds harnesses near-infrared (NIR) light for precise PROTAC release and protein degradation in mouse tumors.


Assuntos
Neoplasias , Animais , Camundongos , Proteólise , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo
7.
bioRxiv ; 2023 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-37066194

RESUMO

Attenuating aberrant transcriptional circuits holds great promise for the treatment of numerous diseases, including cancer. However, development of transcriptional inhibitors is hampered by the lack of a generally accepted functional cellular readout to characterize their target specificity and on-target activity. We benchmarked the direct gene-regulatory signatures of six agents reported as inhibitors of the oncogenic transcription factor MYB against targeted MYB degradation in a nascent transcriptomics assay. The inhibitors demonstrated partial specificity for MYB target genes but displayed significant off-target activity. Unexpectedly, the inhibitors displayed bimodal on-target effects, acting as mixed agonists-antagonists. Our data uncover unforeseen agonist effects of small molecules originally developed as TF inhibitors and argue that rapid-kinetics benchmarking against degron models should be used for functional characterization of transcriptional modulators.

8.
Chembiochem ; 24(19): e202300141, 2023 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-37088717

RESUMO

Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.


Assuntos
Neoplasias , Quimera de Direcionamento de Proteólise , Camundongos , Animais , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias/tratamento farmacológico , Transdução de Sinais , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química
9.
Blood ; 141(23): 2841-2852, 2023 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-36877894

RESUMO

Therapeutic targeting of CDK7 has proven beneficial in preclinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in cells from patients with multiple myeloma (MM); its selective targeting counteracts E2F activity via perturbation of the cyclin-dependent kinases/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small-molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression, increasing survival in several mouse models of MM including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use.


Assuntos
Quinase Ativadora de Quinase Dependente de Ciclina , Mieloma Múltiplo , Animais , Camundongos , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Mieloma Múltiplo/genética
10.
J Med Chem ; 66(7): 4617-4632, 2023 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-36946421

RESUMO

Transcriptional enhanced associate domain (TEAD) proteins together with their transcriptional coactivator yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) are important transcription factors and cofactors that regulate gene expression in the Hippo pathway. In mammals, the TEAD families have four homologues: TEAD1 (TEF-1), TEAD2 (TEF-4), TEAD3 (TEF-5), and TEAD4 (TEF-3). Aberrant expression and hyperactivation of TEAD/YAP signaling have been implicated in a variety of malignancies. Recently, TEADs were recognized as being palmitoylated in cells, and the lipophilic palmitate pocket has been successfully targeted by both covalent and noncovalent ligands. In this report, we present the medicinal chemistry effort to develop MYF-03-176 (compound 22) as a selective, cysteine-covalent TEAD inhibitor. MYF-03-176 (compound 22) significantly inhibits TEAD-regulated gene expression and proliferation of the cell lines with TEAD dependence including those derived from mesothelioma and liposarcoma.


Assuntos
Proteínas de Ligação a DNA , Neoplasias , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Transdução de Sinais , Via de Sinalização Hippo , Mamíferos/metabolismo , Fatores de Transcrição de Domínio TEA
11.
bioRxiv ; 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36993171

RESUMO

Lineage-defining transcription factors form densely interconnected circuits in chromatin occupancy assays, but the functional significance of these networks remains underexplored. We reconstructed the functional topology of a leukemia cell transcription network from the direct gene-regulatory programs of eight core transcriptional regulators established in pre-steady state assays coupling targeted protein degradation with nascent transcriptomics. The core regulators displayed narrow, largely non-overlapping direct transcriptional programs, forming a sparsely interconnected functional hierarchy stabilized by incoherent feed-forward loops. BET bromodomain and CDK7 inhibitors disrupted the core regulators' direct programs, acting as mixed agonists/antagonists. The network is predictive of dynamic gene expression behaviors in time-resolved assays and clinically relevant pathway activity in patient populations.

12.
Methods Enzymol ; 681: 1-22, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36764753

RESUMO

The dTAG system is a versatile strategy for tunable control of protein abundance and facilitates the time-resolved assessment of disease-associated protein function. A "co-opted" fusion-based degron peptide, the "dTAG" facilitates the study of endogenous protein function when knocked-in at the endogenous genetic loci of proteins of interest. We combine CRISPR/Cas9 mediated induction of double-strand breaks (DSB) with the delivery of a single-stranded DNA HDR-donor-template via crude preparations of recombinant adeno-associated virus (rAAV). Our approach to knock-in of large (1-2kb) DNA fragments via crude-rAAV mediated HDR donor delivery is rapid and inexpensive. It facilitates genetic modification of a variety of human as well as mouse cell lines at high efficiency and precision.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Camundongos , Humanos , DNA , DNA de Cadeia Simples , Reparo de DNA por Recombinação
14.
Cell Rep ; 41(9): 111727, 2022 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-36450243

RESUMO

Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAPII) at the transcriptional start site (TSS), and induces the promoter-proximal pausing of RNAPII in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAPII pausing and its function in cancer.


Assuntos
Leucemia , Humanos , Metabolômica , Regulação para Baixo , Reparo do DNA , RNA Polimerase II , Heme , Histona-Lisina N-Metiltransferase/genética
15.
Cell Chem Biol ; 29(10): 1470-1481.e31, 2022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36070758

RESUMO

Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of proteins of interest, inducing ubiquitination-dependent degradation. A major bottleneck in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the Kelch-like ECH-associated protein 1 (KEAP1) inhibitor KI696 into a recruitment handle for several targets. While we were able to generate KEAP1-recruiting degraders of BET family and murine focal adhesion kinase (FAK), we discovered that the target scope of KEAP1 was narrow, as targets easily degraded using a cereblon (CRBN)-recruiting degrader were refractory to KEAP1-mediated degradation. Linking the KEAP1-binding ligand to a CRBN-binding ligand resulted in a molecule that induced degradation of KEAP1 but not CRBN. In sum, we characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.


Assuntos
Fator 2 Relacionado a NF-E2 , Ubiquitina-Proteína Ligases , Camundongos , Animais , Ubiquitina-Proteína Ligases/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Ligantes , Fator 2 Relacionado a NF-E2/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Ubiquitinas/metabolismo
16.
Cell Chem Biol ; 29(8): 1273-1287.e8, 2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-35839780

RESUMO

Reactivation of fetal hemoglobin expression by the downregulation of BCL11A is a promising treatment for ß-hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of γ-globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR-Cas9 gene editing. We leveraged the dTAG PROTAC degradation platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by nascent transcriptomics, proteomics, chromatin accessibility, and histone profiling. Among 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in <2 h. Robust HBG1/2 reactivation upon acute BCL11A depletion occurred without the loss of promoter 5-methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci.


Assuntos
Proteínas Nucleares , Proteoma , Proteínas de Transporte/metabolismo , Cromatina/genética , Cromatina/metabolismo , Células Eritroides/metabolismo , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo
17.
Eur J Med Chem ; 238: 114433, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35597007

RESUMO

Cyclin-dependent kinases (CDK) are attractive targets for drug discovery due to their wide range of cellular functions. CDK11 is an understudied CDK with roles in transcription and splicing, cell cycle regulation, neuronal function, and apoptosis. In this study, we describe a medicinal chemistry campaign to identify a CDK11 inhibitor. Employing a promising but nonselective CDK11-targeting scaffold (JWD-047), extensive structure-guided medicinal chemistry modifications led to the identification of ZNL-05-044. A combination of biochemical evaluations and NanoBRET cellular assays for target engagement guided the SAR towards a 2,4-diaminothiazoles CDK11 probe with significantly improved kinome-wide selectivity over JWD-047. CDK11 inhibition with ZNL-05-044 leads to G2/M cell cycle arrest, consistent with prior work evaluating OTS964, and impacts CDK11-dependent mRNA splicing in cells. Together, ZNL-05-044 serves as a tool compound for further optimization and interrogation of the consequences of CDK11 inhibition.


Assuntos
Apoptose , Quinases Ciclina-Dependentes , Pontos de Checagem do Ciclo Celular , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Relação Estrutura-Atividade
18.
Cancer Discov ; 12(7): 1760-1781, 2022 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-35405016

RESUMO

Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To this end, we applied genome-wide screens to discover genetic vulnerabilities in acute myeloid leukemia (AML) cells implicated in inflammatory pathways. We identified the immune modulator IRF2BP2 as a selective AML dependency. We validated AML cell dependency on IRF2BP2 with genetic and protein degradation approaches in vitro and genetically in vivo. Chromatin and global gene-expression studies demonstrated that IRF2BP2 represses IL1ß/TNFα signaling via NFκB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival. SIGNIFICANCE: This study exploits inflammatory programs inherent to AML blasts to identify genetic vulnerabilities in this disease. In doing so, we determined that AML cells are dependent on the transcriptional repressive activity of IRF2BP2 for their survival, revealing cell-intrinsic inflammation as a mechanism priming leukemic blasts for regulated cell death. See related commentary by Puissant and Medyouf, p. 1617. This article is highlighted in the In This Issue feature, p. 1599.


Assuntos
Leucemia Mieloide Aguda , Humanos , Inflamação/genética , Leucemia Mieloide Aguda/genética , NF-kappa B/metabolismo , Transdução de Sinais
19.
Mol Cell ; 82(6): 1140-1155.e11, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35245435

RESUMO

MLL rearrangements produce fusion oncoproteins that drive leukemia development, but the direct effects of MLL-fusion inactivation remain poorly defined. We designed models with degradable MLL::AF9 where treatment with small molecules induces rapid degradation. We leveraged the kinetics of this system to identify a core subset of MLL::AF9 target genes where MLL::AF9 degradation induces changes in transcriptional elongation within 15 minutes. MLL::AF9 degradation subsequently causes loss of a transcriptionally active chromatin landscape. We used this insight to assess the effectiveness of small molecules that target members of the MLL::AF9 multiprotein complex, specifically DOT1L and MENIN. Combined DOT1L/MENIN inhibition resembles MLL::AF9 degradation, whereas single-agent treatment has more modest effects on MLL::AF9 occupancy and gene expression. Our data show that MLL::AF9 degradation leads to decreases in transcriptional elongation prior to changes in chromatin landscape at select loci and that combined inhibition of chromatin complexes releases the MLL::AF9 oncoprotein from chromatin globally.


Assuntos
Leucemia , Proteína de Leucina Linfoide-Mieloide , Cromatina/genética , Humanos , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição/genética
20.
Genes Dev ; 36(5-6): 368-389, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35301220

RESUMO

Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and superresolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes.


Assuntos
Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Rearranjo Gênico , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Oncogenes/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...