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1.
J Biol Chem ; 300(1): 105501, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38016516

RESUMO

Inhibition of cyclin-dependent kinases (CDKs) has evolved as an emerging anticancer strategy. In addition to the cell cycle-regulating CDKs, the transcriptional kinases Cdk12 and Cdk13 have become the focus of interest as they mediate a variety of functions, including the transition from transcription initiation to elongation and termination, precursor mRNA splicing, and intronic polyadenylation. Here, we determine the crystal structure of the small molecular inhibitor SR-4835 bound to the Cdk12/cyclin K complex at 2.68 Å resolution. The compound's benzimidazole moiety is embedded in a unique hydrogen bond network mediated by the kinase hinge region with flanking hydroxy groups of the Y815 and D819 side chains. Whereas the SR-4835 head group targets the adenine-binding pocket, the kinase's glycine-rich loop is shifted down toward the activation loop. Additionally, the αC-helix adopts an inward conformation, and the phosphorylated T-loop threonine interacts with all three canonical arginines, a hallmark of CDK activation that is altered in Cdk12 and Cdk13. Dose-response inhibition measurements with recombinant CMGC kinases show that SR-4835 is highly specific for Cdk12 and Cdk13 following a 10-fold lower potency for Cdk10. Whereas other CDK-targeting compounds exhibit tighter binding affinities and higher potencies for kinase inhibition, SR-4835 can be considered a selective transcription elongation antagonist. Our results provide the basis for a rational improvement of SR-4835 toward Cdk12 inhibition and a gain in selectivity over other transcription regulating CDKs.


Assuntos
Quinases Ciclina-Dependentes , Ciclinas , Poliadenilação , Ciclinas/metabolismo , Conformação Molecular , Humanos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química
2.
Cancers (Basel) ; 14(7)2022 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-35406461

RESUMO

Type II testicular germ cell tumors (TGCT) are the most frequently diagnosed solid malignancy in young men. Up to 15% of patients with metastatic non-seminomas show cisplatin resistance and a very poor survival rate due to lacking treatment options. Transcriptional cyclin-dependent kinases (CDK) have been shown to be effective targets in the treatment of different types of cancer. Here, we investigated the effects of the CDK inhibitors dinaciclib, flavopiridol, YKL-5-124, THZ1, NVP2, SY0351 and THZ531. An XTT viability assay revealed a strong cytotoxic impact of CDK7/12/13 inhibitor SY0351 and CDK9 inhibitor NVP2 on the TGCT wild-type cell lines (2102EP, NCCIT, TCam2) and the cisplatin-resistant cell lines (2102EP-R, NCCIT-R). The CDK7 inhibitor YKL-5-124 showed a strong impact on 2102EP, 2102EP-R, NCCIT and NCCIT-R cell lines, leaving the MPAF control cell line mostly unaffected. FACS-based analysis revealed mild effects on the cell cycle of 2102EP and TCam2 cells after SY0351, YKL-5-124 or NVP2 treatment. Molecular analysis showed a cell-line-specific response for SY0351 and NVP2 inhibition while YKL-5-124 induced similar molecular changes in 2102EP, TCam2 and MPAF cells. Thus, after TGCT subtype determination, CDK inhibitors might be a potential alternative for optimized and individualized therapy independent of chemotherapy sensitivity.

3.
Open Biol ; 12(3): 210381, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35291876

RESUMO

Cyclin-dependent kinases (CDKs) are key players in cell cycle regulation and transcription. The CDK-family member Cdk10 is important for neural development and can act as a tumour suppressor, but the underlying molecular mechanisms are largely unknown. Here, we provide an in-depth analysis of Cdk10 substrate specificity and function. Using recombinant Cdk10/CycQ protein complexes, we characterize RNA pol II CTD, c-MYC and RB1 as in vitro protein substrates. Using an analogue-sensitive mutant kinase, we identify 89 different Cdk10 phosphosites in HEK cells originating from 66 different proteins. Among these, proteins involved in cell cycle, translation, stress response, growth signalling, as well as rRNA, and mRNA transcriptional regulation, are found. Of a set of pan-selective CDK- and Cdk9-specific inhibitors tested, all inhibited Cdk10/CycQ at least five times weaker than their proposed target kinases. We also identify Cdk10 as an in vitro substrate of Cdk1 and Cdk5 at multiple sites, allowing for a potential cross-talk between these CDKs. With this functional characterization, Cdk10 adopts a hybrid position in both cell cycle and transcriptional regulation.


Assuntos
Quinases Ciclina-Dependentes , Ciclinas/metabolismo , RNA Polimerase II , Ciclo Celular , Divisão Celular , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Humanos , RNA Polimerase II/metabolismo
4.
Nat Commun ; 12(1): 6607, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34785661

RESUMO

Homeodomain-interacting protein kinases (HIPKs) belong to the CMGC kinase family and are closely related to dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). HIPKs are regulators of various signaling pathways and involved in the pathology of cancer, chronic fibrosis, diabetes, and multiple neurodegenerative diseases. Here, we report the crystal structure of HIPK3 in its apo form at 2.5 Å resolution. Recombinant HIPKs and DYRK1A are auto-activated and phosphorylate the negative elongation factor SPT5, the transcription factor c-Myc, and the C-terminal domain of RNA polymerase II, suggesting a direct function in transcriptional regulation. Based on a database search, we identified abemaciclib, an FDA-approved Cdk4/Cdk6 inhibitor used for the treatment of metastatic breast cancer, as potent inhibitor of HIPK2, HIPK3, and DYRK1A. We determined the crystal structures of HIPK3 and DYRK1A bound to abemaciclib, showing a similar binding mode to the hinge region of the kinase as observed for Cdk6. Remarkably, DYRK1A is inhibited by abemaciclib to the same extent as Cdk4/Cdk6 in vitro, raising the question of whether targeting of DYRK1A contributes to the transcriptional inhibition and therapeutic activity of abemaciclib.


Assuntos
Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteínas Ribossômicas/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proteínas de Transporte , Cristalografia por Raios X , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases Dyrk
5.
Mol Cell ; 74(4): 674-687.e11, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30928206

RESUMO

The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.


Assuntos
Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinases Ciclina-Dependentes/genética , Chaperonas de Histonas/genética , Humanos , Neoplasias/genética , Regiões Promotoras Genéticas , Quinase Ativadora de Quinase Dependente de Ciclina
6.
Cell Chem Biol ; 26(6): 792-803.e10, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30905681

RESUMO

Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G1/S transition and inhibition of E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may be useful for treatment of cancers marked by E2F misregulation.


Assuntos
Ciclo Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirróis/farmacologia , Ciclo Celular/genética , Linhagem Celular , Quinases Ciclina-Dependentes/metabolismo , Humanos , Células Jurkat , Masculino , Fenótipo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirróis/química , Quinase Ativadora de Quinase Dependente de Ciclina
7.
Curr Neuropharmacol ; 12(1): 37-43, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24533014

RESUMO

The purines ATP and adenosine are widely recognized for their neuromodulatory effects. They have been shown to have effects on neurons via various receptors and interactions with glial cells. In particular, long-term potentiation (LTP) in hippocampal slice preparations has been found to be modulated by ATP and adenosine. This review gives an overview of purinergic signaling in relation to hippocampal LTP and memory formation. The data supports the hypothesis that adenosine mediates a tonic suppression of synaptic transmission. Thus, low adenosine levels appear to increase basal synaptic activity via a decreased activation of the inhibitor A1 receptor, consequently making it more difficult to induce LTP because of lower contrast. During high stimulation, the inhibition of neighboring pathways by adenosine, in combination with an A2a receptor activation, appears to increase contrast of excited pathways against a nonexcited background. This would enable amplification of specific signaling while suppressing non-specific events. Although a clear role for purinergic signaling in LTP is evident, more studies are needed to scrutinize the modulatory role of ATP and adenosine and their receptors in synaptic plasticity and memory.

8.
Biochem Biophys Res Commun ; 412(4): 606-11, 2011 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-21855532

RESUMO

Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria.


Assuntos
Metilação de DNA/efeitos dos fármacos , Depsipeptídeos/farmacologia , Inativação Gênica/efeitos dos fármacos , Glutationa S-Transferase pi/biossíntese , Inibidores de Histona Desacetilases/farmacologia , Neoplasias da Próstata/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Glutationa S-Transferase pi/genética , Humanos , Masculino , Neoplasias da Próstata/enzimologia
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